Method for Detecting the Purity and Related Substances of Amifostine Intermediate by High Performance Liquid Chromatography

Abstract

The present invention belongs to the field of pharmaceutical analysis and detection, and relates to a method for detecting the purity of amifostine intermediate M2 and its related substances. The chromatographic conditions are a chromatographic column filled with unbonded silica gel particles; acetonitrile is used as mobile phase A, and a 50 mmol / L ammonium formate solution adjusted to pH 2.2 with formic acid is used as mobile phase B, and mobile phases A and B are mixed in a certain proportion for gradient elution. The detection method provided by the present invention can quickly and accurately detect the purity of amifostine intermediate M2 and the content of its related substances. In particular, the content of the genotoxic impurity M2-A can be monitored in intermediate M2, thereby indicating that there may be a safety risk in intermediate M2 compound, and it is necessary to effectively control the content of genotoxic impurity M2-A in the intermediate process; thus, it can ensure that the content of genotoxic impurity M2-A flowing into the amifostine API in the subsequent reaction meets the quality standards, which is of great significance for the synthesis and quality control of the final product amifostine.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis and detection, and particularly relates to a method for detecting the purity and related substances of amifostine intermediate by high performance liquid chromatography. Background Art

[0002] Amifostine, an organic sulfur-phosphorus compound cytoprotectant developed by CLINIGEN, is hydrolyzed and dephosphorylated by alkaline phosphatase in tissues and metabolized into a thiol-containing active metabolite (H2N-(CH2)3-NH-(CH2)2-SH). Since the thiol group has the function of scavenging free radicals in tissues, it can reduce the toxicity of cisplatin, cyclophosphamide, mitomycin, etc. Amifostine is a broad-spectrum normal cell protectant and is mainly used for the adjuvant treatment of various cancers. It can reduce the toxicity of chemotherapy to the kidneys, bone marrow, heart, ears and nervous system, and significantly reduce the occurrence of oral dryness and mucositis when used before radiotherapy.

[0003] In the amifostine synthesis methods disclosed in the prior art, N-(2-bromoethyl)-1,3-propanediamine dihydrobromide (abbreviated as M2) is usually used as an important intermediate in the amifostine synthesis process. Its molecular formula is C5H 15 Br3N2, with a molecular weight of 342.90, and its chemical structure is as follows:

[0004]

[0005] Since intermediate M2 is the previous step in the synthesis of amifostine product, the purity and impurities of intermediate M2 directly affect the quality of amifostine, and thus directly affect the safety and efficacy of the drug. Therefore, monitoring the purity of intermediate M2 and the content of its impurities in the intermediate M2 process plays a very important role in the quality control of amifostine.

[0006] In the synthesis process of amifostine, there may be key by-products and impurities remaining from the reaction raw materials in intermediate M2. There are three related substances controlled in the synthesis process of amifostine intermediate M2, which are as follows:

[0007] Intermediate M1, N-(2-hydroxyethyl)-1,3-propanediamine dihydrobromide, an impurity remaining from the reaction raw material, and its chemical structure is as follows:

[0008]

[0009] Homopiperazine, 1,4-diazacycloheptane, a by-product, and its chemical structure is as follows:

[0010]

[0011] M2-A, N,N-bis(2-bromoethyl)-1,3-propanediamine dihydrobromide, a side reaction product, has the chemical structure as shown in the following formula:

[0012]

[0013] For the above-mentioned impurities, especially the M2-A impurity, its structure contains an alkyl halide structure, which is a warning structure. Therefore, M2-A is a potential genotoxic impurity. According to the ICH M7 guideline, an impurity with a warning structure, unrelated to the structure of the drug substance, and without mutagenicity data is judged as a Class 3 mutagenic impurity, and the content of this type of impurity should be controlled below the acceptable limit (appropriate TTC, where TTC refers to the threshold of toxicological concern for development, used to determine the acceptable intake for the risk of no carcinogenic or other toxic effects of all unstudied chemical substances).

[0014] Since amifostine is applicable to the treatment of ovarian cancer, radiotherapy for head and neck cancer, and non-small cell lung cancer, and the treatment cycles are all less than 10 years, according to the requirements of the ICH M7 guideline, for amifostine used within 10 years, the acceptable intake of TTC for impurities is 10 μg / day. The dosage specified in the original research product instruction is 910 mg / m 2 , combined with the bacterial endotoxin guideline in the European Pharmacopoeia, the maximum human skin surface area is 1.8 m 2 , and it is calculated that the maximum daily dose of API is 1.638 g / day. Then the safety limit of M2-A in amifostine is (10 μg / day) / (1.638 g / day) = 6 ppm.

[0015] After retrieval, there is no report on a method for separating the above-mentioned known impurity detection method yet. However, there is a literature ("Research on New Analytical Methods of Amifostine and Its Intermediates", a master's thesis of Dalian University of Technology in 2014) reporting that the chromatographic column used is Phenomenex Luna C18 (4.6 mm × 250 mm, 5 μm), with methanol - 0.94 g / L sodium hexanesulfonate aqueous solution (pH adjusted to 2.5 with phosphoric acid) at a ratio of 40:60, a diode array detector, a detection wavelength of 220 nm, a flow rate of 1 ml / min, and an injection volume of 10 μl for the reverse high-performance liquid chromatography conditions, and an analytical method for intermediate M2 was established. The inventor of the present invention tried to use the above chromatographic conditions and adjusted the ratio of the mobile phase methanol - 0.94 g / L sodium hexanesulfonate aqueous solution (pH adjusted to 2.5 with phosphoric acid) to 5:95, and detected the purity of intermediate M2 and its related substances. It was found that there was only one peak time. After analysis, M2-A is similar in structure to M2, and its free form has no ultraviolet-absorbing functional groups. It is speculated that it is the peak time of bromide ions. The inventor confirmed the above results in Comparative Example 1. Therefore, it shows that the literature actually calculates the content of M2 indirectly based on the bromide ion content by detecting the bromide ion content in intermediate M2. This method is not applicable to the detection of the purity of intermediate M2 and its related substances.

[0016] Therefore, there is an urgent need for a method that can not only quickly, simply and accurately detect the purity of intermediate M2, but also detect the content of genotoxic impurity M2-A and other related substances in intermediate M2. Summary of the Invention

[0017] The first object of the present invention is to provide a method for quickly and accurately detecting the purity of intermediate M2.

[0018] The second object of the present invention is to provide a method for monitoring the content of genotoxic impurity M2-A in intermediate M2, so as to indicate that there may be safety risks in intermediate M2 compounds, and it is necessary to effectively control the content of genotoxic impurity M2-A in the intermediate process, and further ensure that the residue of genotoxic impurity M2-A in the final product amifostine meets the quality requirements.

[0019] The third object of the present invention is to provide a method that can not only detect the content of genotoxic impurity M2-A in intermediate M2, but also detect the content of other multiple unknown and / or known related substances at the same time.

[0020] To achieve the above object, the present invention provides a method for determining the purity of the intermediate of amifostine, N-(2-bromoethyl)-1,3-propanediamine dihydrobromide and its related substances by high performance liquid chromatography. The high performance liquid chromatography uses a chromatographic column filled with unbonded silica gel particles. Mobile phase A is acetonitrile, and mobile phase B is a 50 ± 5 mmol / L ammonium formate solution with the pH adjusted to 2.2 ± 0.1 with formic acid. Mobile phases A and B are subjected to gradient elution in a certain proportion;

[0021] Further, the gradient elution ratio of the present invention is: 0 - 2 min, 80% A; 2 - 30 min, 80% → 50% A; 30 - 30.1 min, 50% → 80% A; 30.1 - 35 min, 80% A;

[0022] Further, the chromatographic column of the present invention is Waters Altantis HILIC Slica, specifications: 4.6 mm × 250 mm × 5 μm;

[0023] Further, mobile phase B of the present invention is a 50 mmol / L ammonium formate solution with the pH adjusted to 2.2 with formic acid.

[0024] Further, the flow rate of the mobile phase of the present invention is 0.8 - 1.2 mL / min, preferably 1 mL / min;

[0025] Further, the injection volume of the high performance liquid chromatography of the present invention is 10 - 20 μl, preferably 10 μl;

[0026] Further, the column temperature of the high performance liquid chromatography of the present invention is 30 - 40 °C, preferably 35 °C;

[0027] Further, the detector of the high performance liquid chromatography of the present invention is CAD;

[0028] Furthermore, the specific chromatographic conditions of the high performance liquid chromatography of the present invention are: using a Waters Altantis HILIC Slica chromatographic column with specifications of 4.6 mm × 250 mm, 5 μm; using acetonitrile as mobile phase A and a 50 mmol / L ammonium formate solution (with the pH adjusted to 2.2 with formic acid) as mobile phase B; the flow rate is 1 ml / min; the detector is CAD; the column temperature is 35 °C; the injection volume is 10 μl, and the gradient elution program is: 0 - 2 min, 80% A; 2 - 30 min, 80% → 50% A; 30 - 30.1 min, 50% → 80% A; 30.1 - 35 min, 80% A;

[0029] Furthermore, the related substances in the present invention are one or more of N-(2-hydroxyethyl)-1,3-propanediamine dihydrobromide, N,N-bis(2-bromoethyl)-1,3-propanediamine dihydrobromide, and homopiperazine;

[0030] Furthermore, the present invention also includes the preparation of the test solution, specifically prepared as follows: Weigh an appropriate amount of the amifostine intermediate, place it in a volumetric flask, dissolve it with a solvent and dilute it to a solution containing about 5 mg of the amifostine intermediate per 1 ml;

[0031] Furthermore, the solvent in the present invention is a mixed solution of mobile phase A and B with a volume ratio of 80:20;

[0032] Furthermore, the detection method in the present invention is used to detect the purity of the amifostine intermediate;

[0033] Furthermore, the detection method in the present invention is used to detect the content of genotoxic impurities and other unknown and / or known impurities in the amifostine intermediate.

[0034] The present invention also provides a compound having the structure shown in formula (I):

[0035]

[0036] Furthermore, the compound shown in formula (I) provided by the present invention is used as a reference standard or control in the detection of related substances of amifostine or its intermediate N-(2-bromoethyl)-1,3-propanediamine dihydrobromide.

[0037] According to some specific embodiments of the present invention, the detection method includes the following steps:

[0038] (1) Blank solution / solvent / diluent: A mixed solution of mobile phase A and mobile phase B with a volume ratio of 80:20;

[0039] (2) Localization solution: Weigh an appropriate amount of the M1 impurity sample, place it in a 10 ml volumetric flask, add the solvent prepared in (1) to dissolve and dilute to the scale, shake well, and obtain the M1 impurity localization solution; Weigh an appropriate amount of the M2-A impurity sample, place it in a 10 ml volumetric flask, add the solvent prepared in (1) to dissolve and dilute to the scale, shake well, and obtain the M2-A impurity localization solution;

[0040] (3) Test solution: Weigh an appropriate amount of the amifostine intermediate M2, place it in a volumetric flask, dissolve it with the solvent prepared in (1) and dilute it to a solution containing about 5 mg of the amifostine intermediate per 1 ml;

[0041] (4) Set the chromatographic conditions: Use a Waters Altantis HILIC Slica chromatographic column with a specification of 4.6 mm × 250 mm and 5 μm particle size; use acetonitrile as mobile phase A and 50 mmol / L ammonium formate solution (adjusted to pH 2.2 with formic acid) as mobile phase B; the flow rate is 1 ml / min; the detector is CAD; the column temperature is 35 °C; the injection volume is 10 μl, and the gradient elution program is as follows: 0 - 2 min, 80% A; 2 - 30 min, 80% → 50% A; 30 - 30.1 min, 50% → 80% A; 30.1 - 35 min, 80% A;

[0042] (5) Take 10 μl of the solutions from steps (1), (2), and (3) respectively, inject them into the liquid chromatograph, and record the chromatograms; calculate the purity of amifostine intermediate M2 and the content of its related substances according to the area normalization method.

[0043] The present invention has the following beneficial effects:

[0044] The present invention provides a method for detecting amifostine intermediate M2, which can not only effectively separate intermediate M2 and its related substances, but also qualitatively and quantitatively analyze the purity of intermediate M2 and its related substances. This method has the advantages of high sensitivity, good repeatability, specificity, and durability.

[0045] The detection method provided by the present invention can quickly and accurately detect the content of genotoxic impurity M2-A in amifostine intermediate M2, thereby indicating the possible safety risks of intermediate M2 compounds and guiding the further impurity safety evaluation and control.

[0046] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can be made.

[0047] The above content of the present invention will be further described in detail below through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0048] Figure 1 High performance liquid chromatogram of screening case one.

[0049] Figure 2 High performance liquid chromatogram of screening case two.

[0050] Figure 3 High performance liquid chromatogram of screening case three.

[0051] Figure 4 High performance liquid chromatography (HPLC) chromatogram of Screening Case 4.

[0052] Figure 5 High performance liquid chromatography (HPLC) chromatogram of Screening Case 5.

[0053] Figure 6 High performance liquid chromatography (HPLC) chromatogram of Screening Case 6.

[0054] Figure 7 High performance liquid chromatography (HPLC) chromatogram of system suitability solution.

[0055] Figure 8 High performance liquid chromatography (HPLC) chromatogram of Test Sample A.

[0056] Figure 9 High performance liquid chromatography (HPLC) chromatogram of Test Sample B.

[0057] Figure 10 High performance liquid chromatography (HPLC) chromatogram of Test Sample C.

[0058] Figure 11 High performance liquid chromatography (HPLC) chromatogram of amifostine test sample solution.

[0059] Figure 12 High performance liquid chromatography (HPLC) chromatogram of Test Sample Solution of Intermediate M2.

[0060] Figure 13 High performance liquid chromatography (HPLC) chromatogram of sodium bromide localization solution. Detailed implementation manners

[0061] In order to make the technical solutions described in the present invention clearer and more understandable to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0062] In the following examples, the raw materials, reagents or devices used can be obtained from conventional commercial channels or can be obtained by existing known methods unless otherwise specified.

[0063] In the present invention, the abbreviations and technical terms involved have the following meanings:

[0064] CAD: Electrospray detector.

[0065] Preparation method of M2-A: Add 40.36 g of N,N'-bis(2-hydroxyethyl)-1,3-propanediamine hydrobromide and 240 ml of sulfolane to a three-necked flask, stir and heat to 110 °C, dropwise add 67.31 g of phosphorus tribromide, and keep the reaction at this temperature for 2 h. After cooling, a large amount of solid appears. Add a small amount of ethyl acetate dropwise and then filter to obtain the solid. Heat the solid with 240 ml of methanol to reflux. After complete dissolution and clarification, cool to crystallize, filter and dry to obtain the solid M2-A. The structure identification data are as follows:

[0066] 1 H-NMR (400 HZ, DMSO-d6): δ 2.09 - 2.20 (m, 2H), δ 3.17 - 3.21 (t, 4H), δ 3.51 - 3.54 (t, 4H), δ 3.64 - 3.67 (t, 4H),

[0067] 13 C-NMR (400 HZ, DMSO-d6): δ 22.34, δ 25.85, δ 44.39, δ 48.81,

[0068] Free state MS (m / z): 288.9 [M + H] + 。

[0069] Area normalization method: According to the regulations under each variety, prepare the test solution, inject a certain amount, record the chromatogram. Measure the areas of each peak and the total chromatogram peak area on the chromatogram excluding the solvent peak and the hydrobromic acid peak, and calculate the percentage of the area of each peak in the total peak area.

[0070] According to the detection method of the present invention, both intermediate M2 and genotoxic impurity M2-A are hydrobromates, and two peaks will appear on the high performance liquid chromatogram. One peak is hydrobromic acid, and the other peak is the free state of intermediate M2 or the free state of genotoxic impurity M2-A. When calculating, determine the purity of the main component M2 based on the peak of the free state of intermediate M2; determine the content of impurity M2-A based on the peak of the free state of genotoxic impurity M2-A.

[0071] In the following examples, N,N-bis(2-bromoethyl)-1,3-propanediamine dihydrobromate is M2-A, and is sometimes labeled as Impurity A in the examples; N-(2-hydroxyethyl)-1,3-propanediamine dihydrobromate in the examples is M1, and is sometimes labeled as SM1 in the examples; hydrobromic acid in the examples is sometimes labeled as HBr and sometimes labeled as Br - 。

[0072] Detection method for the purity of intermediate M2 and its impurities in Example 1

[0073] Blank solution / solvent / diluent: A mixed solution of acetonitrile and 50 mmol / L ammonium formate (adjust the pH value to 2.2 with formic acid) with a volume ratio of 80:20.

[0074] Test solution: Take about 50 mg of amifostine intermediate M2, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with the above solvent, and shake well to obtain.

[0075] Chromatographic conditions: Use a Waters Altantis HILIC Slica (4.6 mm × 250 mm, 5 μm) chromatographic column; use acetonitrile as mobile phase A and 50 mmol / L ammonium formate solution (adjust the pH value to 2.2 with formic acid) as mobile phase B; the flow rate is 1 ml / min; the detector is CAD; the column temperature is 35 °C; the injection volume is 10 μl, and the gradient elution program is as follows: 0 - 2 min, 80% A; 2 - 30 min, 80% → 50% A; 30 - 30.1 min, 50% → 80% A; 30.1 - 35 min, 80% A.

[0076] Determination method: Accurately measure 10 μl each of the blank solution and the test solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0077] Limit: In the chromatogram of the test solution, deduct the chromatographic peak of the blank solvent, and calculate by the area normalization method respectively. The purity of intermediate M2 shall not be less than 94%. Except that the content of genotoxic impurity intermediate M2-A shall not exceed 2%, the content of other maximum single impurities shall also not exceed 1%.

[0078] Research on the purity of intermediate M2 and its impurity detection method in Example 2

[0079] Solution preparation:

[0080] Test solution - 1: Weigh an appropriate amount of intermediate M2 sample, place it in a volumetric flask, dissolve it with 50% acetonitrile solvent and dilute to a solution containing about 5 mg of intermediate M2 per 1 ml.

[0081] Test solution - 2: Weigh an appropriate amount of intermediate M2 sample, place it in a volumetric flask, add 50 mmol / L ammonium formate solution (adjust the pH value to 2.0 with formic acid) to dissolve and dilute to a solution containing about 5 mg of intermediate M2 per 1 ml.

[0082] Homopiperazine solution - 1: Weigh an appropriate amount of homopiperazine sample, place it in a volumetric flask, add 50% acetonitrile to dissolve and dilute to a solution containing about 2 mg of homopiperazine impurity per 1 ml.

[0083] Homopiperazine solution - 2: Weigh an appropriate amount of homopiperazine sample, place it in a volumetric flask, add 50 mmol / L ammonium formate solution (adjust the pH value to 2.0 with formic acid) to dissolve and dilute to a solution containing about 2 mg of homopiperazine impurity per 1 ml.

[0084] M1 impurity solution - 1: Weigh an appropriate amount of M1 impurity sample, place it in a volumetric flask, add 50% acetonitrile to dissolve and dilute to a solution containing about 1 mg of M1 impurity per 1 ml.

[0085] M1 impurity solution - 2: Weigh an appropriate amount of M1 impurity sample, place it in a volumetric flask, add 50 mmol / L ammonium formate solution (adjust the pH value to 2.0 with formic acid) and dilute to a solution containing approximately 1 mg of M1 impurity per 1 ml.

[0086] Mixed solution - 1: Take a little of the M2 - A impurity sample, add 0.6 ml of test solution - 1 and 0.4 ml of homopiperazine solution - 1 respectively, and mix well. Take 0.4 ml of this solution, add 0.4 ml of M1 impurity solution - 1, and mix well to obtain it.

[0087] Mixed solution - 2: Take a little of the M2 - A impurity sample, add 0.6 ml of test solution - 2 and 0.4 ml of homopiperazine solution - 2 respectively, and mix well. Take 0.4 ml of this solution, add 0.4 ml of M1 impurity solution - 2, and mix well to obtain it.

[0088] Mixed solution - 3: Take about 50 mg of intermediate M2, place it in a 10 - ml volumetric flask, add 1 ml each of M1 positioning solution (take about 5 mg of M1 reference substance, dissolve and dilute to 10 ml with a mixed solution of mobile phase A and B with a volume ratio of 80:20), M2 - A positioning solution (take about 5 mg of M2 - A reference substance, dissolve and dilute to 10 ml with a mixed solution of mobile phase A and B with a volume ratio of 80:20), and homopiperazine positioning solution (take about 5 mg of homopiperazine, dissolve and dilute to 10 ml with a mixed solution of mobile phase A and B with a volume ratio of 80:20), dissolve with the solvent (a mixed solution of mobile phase A and B with a volume ratio of 80:20) and dilute to the scale, shake well to obtain it.

[0089] 2.1 Screening case one

[0090] Using Waters Altantis HILIC Slica (4.6 mm × 250 mm, 5 μm) as the chromatographic column, acetonitrile as mobile phase A, 20 mmol / L ammonium formate solution (adjust the pH value to 4.0 with formic acid) as mobile phase B, column temperature 30 °C, detector CAD, injection volume 10 μl, detect mixed solution - 1 according to gradient elution 1 program, and record the chromatogram.

[0091] Gradient elution 1: 0 - 5 min, 80% → 65% A; 5 - 7 min, 65% A; 7 - 10 min, 65% → 20% A; 10 - 20 min, 20% A; 20 - 20.1 min, 20% → 80% A; 20.1 - 25 min, 80% A.

[0092] The chromatogram is shown in Figure 1 , and the results show that: under these conditions, the peak shapes of each component are poor and the resolution is not good.

[0093] 2.2 Screening case two

[0094] On the basis of Screening Case 1, except for changing the pH of mobile phase B to 3.0, other detection conditions remain unchanged. The mixed solution - 1 is detected according to the above gradient elution 1 program, and the chromatogram is recorded.

[0095] The chromatogram is shown in Figure 2 , and the results show that the resolution is relatively better than that of Screening Case 1, and the shapes of some peaks need to be improved.

[0096] 2.3 Screening Case 3

[0097] On the basis of Screening Case 2, except for changing the concentration, pH value of mobile phase B and the gradient elution program, other detection conditions remain unchanged. The specific mobile phase is acetonitrile as mobile phase A, and 50 mmol / L ammonium formate solution (adjusted to pH 2.5 with formic acid) as mobile phase B. The mixed solution - 1 is detected according to the gradient elution 2 program, and the chromatogram is recorded.

[0098] Gradient elution 2: 0 - 3 min, 80% A; 3 - 5 min, 80% → 65% A; 5 - 20 min, 65% → 20% A; 20 - 22 min, 20% A; 22 - 22.1 min, 20% → 80% A; 22.1 - 25 min, 80% A.

[0099] The chromatogram is shown in Figure 3 , and the results show that the retention times of M2, M2 - A, M1 and homopiperazine are 11.030 min, 10.356 min, 11.683 min and 12.027 min respectively. The number of theoretical plates is greater than 10000. The peaks can be basically separated from each other, and the minimum resolution is 1.83. However, the peak of homopiperazine in the mixed solution - 1 is interfered, and there are impurities not separated.

[0100] 2.4 Screening Case 4

[0101] On the basis of Screening Case 3, except for changing the column temperature to 35 °C, other detection conditions remain unchanged. The mixed solution - 1 is detected according to the above gradient elution 2 program, and the chromatogram is recorded.

[0102] The chromatogram is shown in Figure 4 , and the results show that the peaks can be basically separated from each other, the minimum resolution is 2.09, and the separation between the homopiperazine peak and the unknown impurity is slightly improved, but there is still interference.

[0103] 2.5 Screening Case 5

[0104] On the basis of Screening Case 4, except for changing the pH value of mobile phase B and the gradient elution program, other detection conditions remain unchanged. The specific mobile phase is acetonitrile as mobile phase A, and 50 mmol / L ammonium formate solution (adjusted to pH 2.0 with formic acid) as mobile phase B. The mixed solution - 2 is detected according to the gradient elution 3 program, and the chromatogram is recorded.

[0105] Gradient elution 3: from 0 to 3 min, 80% A; from 3 to 10 min, 80% → 50% A; from 10 to 11 min, 50% A; from 11 to 16 min, 50% → 30% A; from 16 to 20 min, 30% → 80% A; from 20 to 30 min, 80% A.

[0106] The chromatogram is shown in Figure 5 , and the results show that: the blank solvent has no interference, the column efficiency of each impurity is good, the peak shape is good, and the peaks can be basically separated from each other, but the resolution between homopiperazine and M1 is not very good (1.46 < 1.5).

[0107] 2.6 Screening case six

[0108] On the basis of screening case five, except for changing the pH value of mobile phase B and the gradient elution program, other detection conditions remain unchanged. Specifically, acetonitrile is used as mobile phase A, and 50 mmol / L ammonium formate solution (pH value adjusted to 2.2 with formic acid) is used as mobile phase B. The mixed solution - 3 is detected according to gradient elution program 4, and the chromatogram is recorded.

[0109] Gradient elution 4: from 0 to 2 min, 80% A; from 2 to 30 min, 80% → 50% A; from 30 to 30.1 min, 50% → 80% A; from 30.1 to 35 min, 80% A.

[0110] The chromatogram is shown in Figure 6 , and the results show that: the blank solvent has no interference with the detection, M2 and each impurity have good retention, good resolution, and good peak shape, and the resolution between homopiperazine and M1 is 2.44.

[0111] Methodology verification of Example 3

[0112] After detecting multiple batches of intermediate M2 samples, homopiperazine impurities were not detected, indicating that there is almost no homopiperazine impurity in intermediate M2. Therefore, the situation of homopiperazine was not investigated in the methodology verification. The methodology specificity, detection limit, repeatability, stability, and durability were further investigated according to the chromatographic conditions of screening case six.

[0113] Blank solution / solvent / diluent: A mixed solution of mobile phase A (acetonitrile) and mobile phase B (50 mmol / L ammonium formate solution with pH value adjusted to 2.2 with formic acid) with a volume ratio of 80:20.

[0114] M1 impurity localization solution: Take about 5 mg of M1 reference substance, place it in a 10 ml volumetric flask, add an appropriate amount of the above solvent to dissolve it and dilute to the scale, shake well, and obtain it.

[0115] M2-A impurity localization solution: Take about 5 mg of M2-A reference substance, place it in a 10-ml volumetric flask, add an appropriate amount of the above solvent to dissolve it and dilute to the mark, shake well, and you will get it.

[0116] System suitability solution: Take about 50 mg of M2, place it in a 10-ml volumetric flask, add 1 ml each of M1 localization solution and M2-A localization solution, dissolve with the above solvent and dilute to the mark, shake well, and you will get it.

[0117] Test solution: Take about 50 mg of M2, place it in a 10-ml volumetric flask, add an appropriate amount of the above solvent to dissolve it and dilute to the mark, shake well, and you will get it.

[0118] 3.1 Specificity

[0119] Inject the M1, M2-A impurity localization solution, system suitability solution and blank solution prepared in Example 3 into the high performance liquid chromatograph respectively, and record the chromatogram.

[0120] The results show that: the blank solution does not interfere with the detection of the M2 main peak and each impurity. In the system suitability solution ( Figure 7 ), the resolution between the M2 main peak and the adjacent peak is 7.57; the resolution between M1 and the adjacent peak is 7.16; the resolution between M2-A and the adjacent peak is 5.45. The resolutions are all greater than 1.5, indicating that the detection method of the present invention has good specificity.

[0121] 3.2 Detection limit

[0122] Precisely measure 1 ml each of the M1 impurity localization solution and M2-A impurity localization solution prepared in Example 3, place them in 50-ml volumetric flasks respectively, dilute to the mark with the solvent prepared in Example 3 respectively, and shake well; gradually dilute until the peak heights of impurities M1 and M2-A are 3 to 10 times the baseline noise as the detection limit, and this is used as the detection limit solution.

[0123] Detect the detection limit solution according to the above chromatographic conditions and record the chromatogram. The results are shown in the following table:

[0124] Table 1 Results of detection limit test

[0125] Name Retention time Peak area Signal-to-noise ratio Ratio equivalent to the concentration of the test substance Detection limit of M1 18.419 min 0.2291 5.8 0.065％ Detection limit of M2-A 13.226 min 0.1920 5.4 0.125％

[0126] The results in Table 1 show that the sensitivity of the detection method of the present invention meets the detection requirements.

[0127] 3.3 Repeatability:

[0128] Weigh about 50 mg of the intermediate M2 test substance, place it in a 10-ml volumetric flask, dissolve it with the solvent prepared in Example 3 and dilute to the mark, shake well, and you will get it. Prepare 6 portions in parallel as test solutions.

[0129] Mixed impurity stock solution: Take 10 ml each of the M1 impurity localization solution and M2-A impurity localization solution prepared in Example 3 above, place them in a 25-ml volumetric flask, add an appropriate amount of the solvent prepared in Example 3 to dissolve and dilute to the mark, shake well, and you will get it.

[0130] Weigh about 50 mg of the test sample of intermediate M2, place it in a 10-ml volumetric flask, add 2.5 ml of the mixed impurity stock solution, dissolve it with the solvent prepared in Example 3 and dilute to the mark, shake well, and you will get it. Prepare 6 portions in parallel as the spiked test sample solutions.

[0131] Detect the above 12 solutions according to the above chromatographic conditions and record the chromatograms. The results are shown in the following table:

[0132] Table 2 Results of repeatability test

[0133]

[0134]

[0135] The results in Table 2 show that the repeatability of the test sample solution and the spiked test sample solution is good.

[0136] 3.4 Stability:

[0137] Respectively place the test sample solutions prepared in Example 3 above in the injector (4 °C), take samples and measure at 1, 2, 3, 4, and 5 h respectively to investigate the stability of the solution. The results are shown in the following table:

[0138] Table 3 Results of stability test

[0139]

[0140] The results in Table 3 show that under the condition of 4 °C, the test sample solution is stable within 4 h (the acceptable range of the ratio of the peak area of M2 in the test sample solution injected at each time point to the peak area at 0 h is 98% - 102%).

[0141] 3.5 Robustness

[0142] Respectively change the flow rate (0.8 ml / min, 1.2 ml / min), column temperature (30 °C or 40 °C), mobile phase pH (2.1 or 2.3), and the concentration of buffer salt (ammonium formate solution concentration 45 mmol or 55 mmol), and the other chromatographic conditions are the same as those in Example 1. Inject the system suitability solution and the test sample solution prepared in Example 3 above into the liquid chromatograph respectively, record the chromatograms, and the test results are shown in Tables 4 - 5.

[0143] Table 4 Results of robustness test for system suitability

[0144]

[0145] Table 5 Results of the robustness test for the test solution

[0146]

[0147] The results in Tables 4 - 5 show that after changing the flow rate, column temperature, pH value of the mobile phase, and buffer salt concentration, there are no significant changes in the detected amount and number of impurities in the test solution; the separation between impurities and the main component in the system suitability solution is good. Thus, it can be seen that fine-tuning of the conditions will not affect the determination of related substances, and the detection method of the present invention has good robustness.

[0148] Example 4 Detection of the amifostine intermediate M2 sample

[0149] Take three batches of amifostine intermediate M2 products, defined as test samples A, B, and C respectively, and detect them using the detection method in Example 1. Then, calculate the purity of intermediate M2 and its impurity content by the area normalization method. The detection results are shown in Tables 6 - 8 and Figures 8 - 10 。

[0150] Table 6 Detection results of test sample A

[0151] Peak name Elution time (min) Peak area Percentage of peak area (%) Resolution Bromide ion 3.54 / / / M2-A 13.250 9705556 0.18 / M2 14.994 5284903210 98.88 4.6 SM1 17.331 15873360 0.30 5.6 Homopiperazine / / / / Maximum single impurity 19.609 34025774 0.64 8.6 Total impurities / 59604690 1.12 /

[0152] Table 7 Detection results of test sample B

[0153]

[0154]

[0155] Table 8 Detection results of test sample C

[0156] Peak name Elution time Peak area Percentage of peak area (%) Resolution Bromide ion 3.54 / / / M2-A 13.246 10217224 0.18 / M2 14.979 5499507514 98.79 4.4 SM1 17.327 20403493 0.37 6.0 Homopiperazine / / / /

[0157] Maximum single impurity 19.565 36800610 0.66 9.5 Total impurities / 67,421,327 1.21 /

[0158] As can be seen from Tables 6 - 8, the detection method of the present invention can quickly and accurately detect the purity of amifostine intermediate M2 and the content of its impurities, and the operation is simple, and complete separation can be achieved. Furthermore, it can indicate the possible safety risks of the intermediate M2 compound, pointing the way for further impurity safety evaluation and control.

[0159] Example 5 Investigation on the content limit of the genotoxic impurity M2 - A in intermediate M2

[0160] Using intermediate M2 as a reactant to synthesize the final product amifostine, in which the genotoxic impurity M2-A in intermediate M2 will flow into the amifostine bulk drug during subsequent reactions. Using intermediate M2 containing different contents of M2-A as the starting material to synthesize amifostine, and determining the content limit of the genotoxic impurity M2-A in intermediate M2 by measuring the content of M2-A impurity in the final reaction product amifostine. The synthesis method is as follows:

[0161]

[0162] Method for detecting the content of M2-A in amifostine:

[0163] Test solution of amifostine: Take about 100 mg of amifostine, weigh accurately, place it in a 10 mL volumetric flask, dissolve it completely with 2 mL of water, add 8 mL of methanol and mix well, filter through a 0.2 μm filter membrane, and take the subsequent filtrate in an injection vial, then it is obtained.

[0164] Stock solution of reference substance: Take about 6 mg of M2-A reference substance, weigh accurately, place it in a 10 mL volumetric flask, dissolve it with 2 mL of water, then dilute it to the scale with methanol and shake well, then it is obtained.

[0165] Reference solution: Take about 100 mg of amifostine, weigh accurately, place it in a 10 mL volumetric flask, dissolve it completely with 2 mL of water, add 100 μL of the stock solution of reference substance to the volumetric flask, mix well, then add 8 mL of methanol and mix well, filter through a 0.2 μm filter membrane, and take the subsequent filtrate in an injection vial, then it is obtained.

[0166] Chromatographic conditions: Use YMC-Triart Diol-HILIC as the chromatographic column, with the specification: 4.6×150 mm, 3 μm. Flow rate: 1.0 mL / min; Injection volume: 10 μL; Column temperature: 40 °C.

[0167] Mobile phase: A mixed solution of methanol and water (containing 0.2% formic acid by volume fraction) with a volume ratio of 80:20.

[0168] Mass spectrometry conditions: Electrospray ionization source (ESI), detection in positive ion MRM mode; Dry gas flow rate: 10 L / min; Nebulizing gas flow rate: 3.0 L / min; Heating gas flow rate: 10 L / min; Desolvation temperature: 526 °C; Interface temperature: 300 °C; Heating block temperature: 400 °C; DL tube temperature: 250 °C; Acquisition time: 10 min.

[0169] Take 10 μL each of the test solution and the reference solution, inject them into the LC-MS, and record the mass spectrometry diagram. Calculate the content of the genotoxic impurity M2-A in amifostine by the external standard method, and the measurement results are as follows in the table:

[0170]

[0171] As can be seen from the above, when the content of genotoxic impurity M2-A in intermediate M2 is controlled below 1.0%, it can ensure that there is no residue of genotoxic impurity M2-A in the final product amifostine; when the content of genotoxic impurity M2-A in intermediate M2 is controlled below 2.0%, it can ensure that the content of impurity M2-A in the final product amifostine meets the standard requirements (<6 ppm).

[0172] Comparative Example 1

[0173] Test solution of M2: Weigh 10.20 mg of intermediate M2 and place it in a 10-ml volumetric flask. Dissolve it with water and dilute it to the mark with water, then shake well to obtain the solution.

[0174] NaBr positioning solution: Weigh 6.11 mg of sodium bromide and place it in a 10-ml volumetric flask. Dissolve it with water and dilute it to the mark, then shake well to obtain the solution.

[0175] The inventors attempted to use the analytical method in the master's thesis "Research on New Analytical Methods for Amifostine and Its Intermediates" by Dalian University of Technology in 2014 to examine the test solution of intermediate M2 of this application.

[0176] Using a Phenomenex Luna C18 (4.6 mm × 250 mm, 5 μm) chromatographic column, isocratic elution was carried out with a mixed solution of methanol - 0.94 g / L sodium hexanesulfonate aqueous solution (pH adjusted to 3.0 with phosphoric acid) in a volume ratio of 5:95 as the mobile phase. A diode array was used as the detector, the detection wavelength was 220 nm, and the flow rate was 1 ml / min. Inject 10 μl of the above-prepared test solution of intermediate M2 into the high-performance liquid chromatograph and record the chromatogram.

[0177] The chromatogram is shown in Figure 12 , and the results show that there is only one main peak retention time (2.006 min). After analysis, M2 in the free state has no ultraviolet-absorbing functional groups, and it is speculated that it is the elution time of bromide ions. To confirm the above speculation, the inventors calculated the concentration of bromide ions in the above-prepared test solution of M2 to be 0.48 mg / ml (M2 weighing / M2 molecular weight * bromine molecular weight * 2 / dilution factor = 10.20 / 342.90 * 79.90 * 2 / 10 = 0.48 mg / ml), and prepared a NaBr positioning solution with the same concentration of bromide ions (sodium bromide weighing / sodium bromide molecular weight * bromine molecular weight / dilution factor = 6.11 / 102.9 * 79.90 / 10 = 0.48 mg / ml). Inject 10 μl of the NaBr positioning solution into the high-performance liquid chromatograph and record the chromatogram. The chromatogram is shown in Figure 13 .

[0178] Statistics Figures 12 - 13 The results are as follows:

[0179] Solution name Retention time of the main peak (min) Peak area of the main peak Test solution of M2 2.006 14751950 NaBr localization solution 2.010 14054889

[0180] Combined with Figures 12 - 13 the content of the above table, it is concluded that the retention time, peak shape, and peak area of the main peak in the test solution of intermediate M2 are basically the same as those of the main peak in the NaBr positioning solution, indicating that the main peak in the test solution of intermediate M2 is actually the peak of bromide ion. Therefore, the detection method disclosed in the prior art cannot detect the purity of intermediate M2 and its impurities.

Claims

1. A detection method for determining related substances of the amifostine intermediate N-(2-bromoethyl)-1,3-propanediamine dihydrobromide by high performance liquid chromatography, characterized in that, The high performance liquid chromatography method uses a chromatographic column filled with unbonded silica gel particles. Mobile phase A is acetonitrile, and mobile phase B is a 50 ± 5 mmol / L ammonium formate solution with the pH adjusted to 2.2 ± 0.1 with formic acid. The mobile phases A and B are subjected to gradient elution in a certain proportion. The gradient elution proportion is as follows: 0 - 2 min, 80% A; 2 - 30 min, 80% → 50% A; 30 - 30.1 min, 50% → 80% A; 30.1 - 35 min, 80% A. The chromatographic column is Waters Altantis HILIC Slica, with specifications: 4.6 mm × 250 mm × 5 μm. The detector is CAD. The related substances are N-(2-hydroxyethyl)-1,3-propanediamine dihydrobromide, N,N-bis(2-bromoethyl)-1,3-propanediamine dihydrobromide, and homopiperazine.

2. The detection method according to claim 1, wherein Mobile phase B is a 50 mmol / L ammonium formate solution with the pH adjusted to 2.2 with formic acid.

3. The detection method according to claim 1, wherein The flow rate of the mobile phase is 0.8 - 1.2 mL / min.

4. The detection method according to claim 3, wherein The flow rate of the mobile phase is 1 mL / min.

5. The detection method according to claim 1, wherein The injection volume of the high performance liquid chromatography method is 10 - 20 μl; the column temperature is 30 - 40 °C.

6. The detection method according to claim 5, wherein The injection volume of the high performance liquid chromatography method is 10 μl; the column temperature is 35 °C.

7. The detection method according to claim 1, wherein It also includes the preparation of the test solution, specifically as follows: Weigh an appropriate amount of the amifostine intermediate, place it in a volumetric flask, and dissolve and dilute it with a mixed solution of mobile phases A and B with a volume ratio of 80:20 to obtain a solution containing about 5 mg of the amifostine intermediate per 1 ml.

8. The detection method according to any one of claims 1 - 7 is used for detecting the purity of the amifostine intermediate and / or the content of related substances in the amifostine intermediate.

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