A high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof

Abstract

The invention discloses a high performance liquid chromatography separation and analysis method of ferric citrate and six kinds of organic acids thereof, i.e., a high performance liquid chromatograph is used, with #imgabs0#AQ-C18 5μm×4.6mm×250mm column and Hydro-RP 80A 4μm×4.6mm×250mm column as chromatographic columns, with aqueous phosphoric acid solution and phosphate buffer solution as mobile phases, and the flow rate of the mobile phase is controlled to be 0.80-1.20mL / min; the chromatographic column temperature is 30°C; the injection volume is 20μL; and the chromatographic separation is carried out under the condition that the detection wavelength is 210nm, and the separation and determination of ferric citrate and six kinds of organic acid impurities thereof are effectively realized. The present invention has good peak shape and no tailing, and the method has the characteristics of high feasibility, simple operation, good separation effect, and low separation cost.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis and relates to pharmaceutical-grade ferric citrate, in particular to a high performance liquid chromatography separation and analysis method of ferric citrate and six organic acids thereof. Background Art

[0002] The English name of ferric citrate is Ferric Citrate Hydrate, its chemical name is ferric citrate hydrate, and its molecular formula is FeC6H5O7+nH2O. Ferric citrate is used to treat chronic kidney disease (CKD). In addition to improving anemia in the body, the drug can also delay renal function decline and improve prognosis by regulating calcium-phosphorus metabolism and hemoglobin levels in CKD patients. Iron-phosphorus binders can reduce the blood phosphorus level of patients with chronic kidney disease, and can also improve kidney function and heart function to a certain extent. Therefore, the study of the separation method of organic acid impurities in ferric citrate has important medicinal and industrial value. The structural formula of ferric citrate is shown below:

[0003]

[0004] According to relevant literature reports, the synthesis of ferric citrate mainly uses ferric chloride as the starting material, reacts with sodium hydroxide to generate ferric hydroxide flocs, and the filtered flocs are then reacted with citric acid monohydrate. After the reaction is complete, an organic solvent is added to precipitate the ferric citrate hydrate. The specific process route is as follows:

[0005] FeCl3·6H2O+3NaOH→Fe(OH)3+3NaCl+6H2O

[0006] Fe(OH)3+C6H8O7·H2O→ferric citrate hydrate

[0007] During the synthesis of ferric citrate, citric acid will decompose into a variety of organic acid impurities. The decomposition process of the organic acid and its specific structural formula involved in the present invention is as follows:

[0008]

[0009] The decomposed organic acids are acetic acid, 1.3-acetonedicarboxylic acid, cis-aconitic acid, trans-aconitic acid, acetoacetic acid, and itaconic acid.

[0010] During the synthesis of ferric citrate, citric acid becomes unstable and decomposes when heated, generating a variety of organic acid impurities that affect the activity of the drug. Therefore, controlling the content of organic acids in ferric citrate is of great significance for drugs to improve renal function decline, improve anemia to a certain extent, and protect the safety of medication for the majority of patients. Through a large number of literature searches, there are currently no relevant literature reports on the separation and analysis of organic acid impurities in ferric citrate. Therefore, it is particularly important for pharmaceutical manufacturers to develop a high-performance liquid phase separation and analysis method for the separation of organic acids in ferric citrate. Summary of the invention

[0011] The technical problem to be solved by the present invention is to provide a high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, and to provide a high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof.

[0012] In order to solve the above problems, the present invention provides a high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, which is separated using the following chromatographic separation conditions:

[0013] The mobile phase flow rate was 0.80-1.20 mL / min; the column temperature was 30°C; the injection volume was 20 μL; the detection wavelength was 210-240 nm; the mobile phase was aqueous phosphoric acid solution and phosphate buffer solution; the column was AQ-C18 5μm×4.6mm×250mm column, Hydro-RP 80A 4μm×4.6mm×250mm column, CAPCELL PAK ADME 3μm×4.6mm×250mm column, Ultimate OAA 5μm 4.6mm×300mm column, Acclaim Mixed-Mode WAX-1 5μm×4.6mm×250mm column.

[0014] Furthermore, the chromatographic column may preferably be a chromatographic column with octadecyl bonded silica as a filler. AQ-C18, the column specifications are 5μm, 4.6mm*250mm; Hydro-RP 80A, the column specifications are 4μm, 4.6mm×250mm.

[0015] Furthermore, the flow rate of the mobile phase may preferably be 1.00 mL / min, the column temperature of the chromatographic column is 30° C., and the detection wavelength is 210 nm.

[0016] Furthermore, the mobile phase may preferably be an aqueous phosphoric acid solution, with the pH adjusted to 2.00 with orthophosphoric acid.

[0017] Furthermore, the mobile phase may preferably be a 40 mmol / L diammonium phosphate buffer solution, with the pH adjusted to 2.00 with orthophosphoric acid.

[0018] Furthermore, the high performance liquid chromatograph used may preferably be Agilent 1260 Infinity II.

[0019] The invention aims at the problems existing in the synthesis technology and provides a high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof. The separation method solves the organic acid impurities existing in the process technology for synthesizing ferric citrate, and the organic acid impurities will affect the quality of the medicine. The method ensures the reliability in the selection of raw materials.

[0020] On the other hand, the present invention provides a high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, using a high performance liquid chromatograph and a chromatographic column with octadecyl bonded silica gel as a filler, the method has good peak shape, high sensitivity, good separation degree, and accurate and reliable results. It can effectively, quickly and sensitively detect ferric citrate and six organic acid impurities thereof, and can also be applied to the quality control of related and content.

[0021] The present invention discloses a high-performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof. High-performance liquid chromatography is used to separate organic acid impurities produced in the process of synthesizing ferric citrate. Since these organic acids are highly polar compounds, they are not easy to be retained in a general C18 chromatographic column. After a large number of attempts and screenings, the method can make the analyte fully act on the bonded phase, and strongly retain under 100% pure water phase conditions. The six impurities can be effectively completely separated under the selected chromatographic conditions, and the quality control problem of ferric citrate can be effectively solved. The equipment requirements of the present invention are not high, and expensive ion chromatography is not required for separation. The liquid phase is universal and easy to obtain, and the feasibility is high, the operation is simple, and the universality is good. It has brought great social significance to industrial production and has strong practicality.

[0022] The invention is a novel analysis technology, and there is no separation method for organic acid in ferric citrate at present. The invention has high sensitivity, good separation degree, reliable analysis method, and is suitable for the relevant information and quality control of ferric citrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a chromatographic separation diagram of ferric citrate and six organic acids thereof in Example 1;

[0024] Figure 2 is a chromatographic separation diagram of ferric citrate and six organic acids thereof in Example 2;

[0025] Figure 3 is a chromatographic separation diagram of ferric citrate and six organic acids thereof in Example 3;

[0026] Figure 4 is a chromatographic separation diagram of ferric citrate and six organic acids thereof in Example 4;

[0027] Figure 5 is a chromatographic separation diagram of ferric citrate and six organic acids thereof in Example 5;

[0028] Figure 6 is a chromatographic separation diagram of ferric citrate and six organic acids thereof in Example 6;

[0029] Figure 7 is the chromatographic separation diagram of the citric acid standard in Example 7;

[0030] Figure 8 This is the chromatographic separation diagram of the citric acid standard in Example 8. DETAILED DESCRIPTION

[0031] In order to make the present invention more clearly understood, the preferred embodiments are described in detail below with reference to the accompanying drawings. The instrument models and manufacturer information used in the embodiments are shown in the following table:

[0032]

[0033] The batch numbers and manufacturer information of the impurity standards used in the examples are shown in the following table:

[0034]

[0035]

[0036] Example 1

[0037] A high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, the steps of which are as follows: Weigh 40 mg of acetic acid standard, 20 mg of 1.3-acetone dicarboxylic acid standard, 20 mg of acetoacetic acid, 20 mg of cis-aconitic acid standard, 20 mg of trans-aconitic acid standard, and 20 mg of itaconic acid standard, accurately weigh them in a 100 mL volumetric flask, dissolve them to scale with water, mix them well, and use them as an impurity reserve solution; then accurately transfer 5.0 mL of the solution to a 100 mL volumetric flask, dilute them to scale with water, mix them well, and use them as an impurity mother solution. Weigh 70 mg of citric acid standard, accurately weigh them in a 50 mL volumetric flask, first add a small amount of water to dissolve them, then add 5.0 mL of impurity mother solution, dilute them to scale with water, mix them well, and use them as a separation solution. That is, a high performance liquid chromatograph is used, and a Hydro-RP 80A 4μm×4.6mm×250mm column with octadecyl bonded silica as a filler is used for separation under the following separation conditions:

[0038] Mobile phase: phosphoric acid aqueous solution, pH = 2.00;

[0039] The mobile phase flow rate was 1.0 mL / min;

[0040] The column temperature was 30°C;

[0041] The injection volume was 20 μL;

[0042] The detection wavelength is 210nm;

[0043] The chromatographic separation results are shown in the following table:

[0044] Sample name RT(min) R Acetic acid 6.065 / 1.3-Acetonedicarboxylic acid 7.802 8.27 Acetoacetic acid 8.944 4.55 Citric Acid 10.180 6.58 Cis-aconitic acid 15.806 13.29 trans-aconitic acid 19.777 8.60 Itaconic acid 28.906 14.68

[0045] It can be seen from the results in the above table that the chromatographic conditions studied in this embodiment can completely separate citric acid and its six organic acid impurities with good separation, meeting the requirements of the Chinese Pharmacopoeia; the chromatographic separation spectrum results are shown in Figure 1 .

[0046] Example 2

[0047] A high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, the steps of which are as follows: Weigh 40 mg of acetic acid standard, 20 mg of 1.3-acetone dicarboxylic acid standard, 20 mg of acetoacetic acid, 20 mg of cis-aconitic acid standard, 20 mg of trans-aconitic acid standard, and 20 mg of itaconic acid standard, accurately weigh them in a 100 mL volumetric flask, dissolve them to scale with water, mix them well, and use them as an impurity reserve solution; then accurately transfer 5.0 mL of the solution to a 100 mL volumetric flask, dilute them to scale with water, mix them well, and use them as an impurity mother solution. Weigh 70 mg of citric acid standard, accurately weigh them in a 50 mL volumetric flask, first add a small amount of water to dissolve them, then add 5.0 mL of impurity mother solution, dilute them to scale with water, mix them well, and use them as a separation solution. That is, a high performance liquid chromatograph is used, and a Hydro-RP 80A 4μm×4.6mm×250mm column with octadecyl bonded silica as a filler is used for separation under the following separation conditions:

[0048] Mobile phase: phosphoric acid aqueous solution, pH = 2.00;

[0049] The mobile phase flow rate was 0.8 mL / min;

[0050] The column temperature was 30°C;

[0051] The injection volume was 20 μL;

[0052] The detection wavelength is 210nm;

[0053] The chromatographic separation results are shown in the following table:

[0054] Sample name RT(min) R Acetic acid 7.457 / 1.3-Acetonedicarboxylic acid 10.064 7.13 Acetoacetic acid 10.904 3.12 Citric Acid 12.352 4.18 Cis-aconitic acid 21.163 16.32 trans-aconitic acid 23.938 4.84 Itaconic acid 34.705 14.49

[0055] It can be seen from the results in the above table that the chromatographic conditions studied in this embodiment can completely separate citric acid and its six organic acid impurities, and the separation degree meets the requirements of the Chinese Pharmacopoeia, but the peak time is relatively long, which needs to be considered from multiple factors such as saving time and mobile phase; the chromatographic separation spectrum results are shown in Figure 2 .

[0056] Example 3

[0057] A high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, the steps of which are as follows: Weigh 40 mg of acetic acid standard, 20 mg of 1.3-acetone dicarboxylic acid standard, 20 mg of acetoacetic acid, 20 mg of cis-aconitic acid standard, 20 mg of trans-aconitic acid standard, and 20 mg of itaconic acid standard, accurately weigh them in a 100 mL volumetric flask, dissolve them to scale with water, mix them well, and use them as an impurity reserve solution; then accurately transfer 5.0 mL of the solution to a 100 mL volumetric flask, dilute them to scale with water, mix them well, and use them as an impurity mother solution. Weigh 70 mg of citric acid standard, accurately weigh them in a 50 mL volumetric flask, first add a small amount of water to dissolve them, then add 5.0 mL of impurity mother solution, dilute them to scale with water, mix them well, and use them as a separation solution. That is, a high performance liquid chromatograph is used, and a Hydro-RP 80A 4μm×4.6mm×250mm column with octadecyl bonded silica as a filler is used for separation under the following separation conditions:

[0058] Mobile phase: phosphoric acid aqueous solution, pH = 2.00;

[0059] The mobile phase flow rate was 1.2 mL / min;

[0060] The column temperature was 30°C;

[0061] The injection volume was 20 μL;

[0062] The detection wavelength is 210nm;

[0063] The chromatographic separation results are shown in the following table:

[0064] Sample name RT(min) R Acetic acid 5.760 / 1.3-Acetonedicarboxylic acid 6.295 2.31 Acetoacetic acid 6.636 1.84 Citric Acid 8.668 7.19 Cis-aconitic acid 11.818 8.95 trans-aconitic acid 14.897 8.26 Itaconic acid 19.459 9.78

[0065] It can be seen from the results in the above table that the chromatographic conditions studied in this example can completely separate citric acid and its six organic acid impurities, and the separation degree meets the requirements of the Chinese Pharmacopoeia, but the retention time of 1,3-acetone dicarboxylic acid and acetoacetic acid is relatively close, and the separation degree is close to the specified limit; the chromatographic separation spectrum results are shown in Figure 3 .

[0066] Example 4

[0067] A high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, the steps of which are as follows: Weigh 40 mg of acetic acid standard, 20 mg of 1.3-acetone dicarboxylic acid standard, 20 mg of acetoacetic acid, 20 mg of cis-aconitic acid standard, 20 mg of trans-aconitic acid standard, and 20 mg of itaconic acid standard, accurately weigh them in a 100 mL volumetric flask, dissolve them to scale with water, mix them well, and use them as an impurity reserve solution; then accurately transfer 5.0 mL of the solution to a 100 mL volumetric flask, dilute them to scale with water, mix them well, and use them as an impurity mother solution. Weigh 70 mg of citric acid standard, accurately weigh them in a 50 mL volumetric flask, first add a small amount of water to dissolve them, then add 5.0 mL of impurity mother solution, dilute them to scale with water, mix them well, and use them as a separation solution. That is, a high performance liquid chromatograph is used, and a Hydro-RP 80A 4μm×4.6mm×250mm column with octadecyl bonded silica as a filler is used for separation under the following separation conditions:

[0068] Mobile phase: 40mmol / L ammonium dihydrogen phosphate aqueous solution, pH = 2.00;

[0069] The mobile phase flow rate was 1.0 mL / min;

[0070] The column temperature was 30°C;

[0071] The injection volume was 20 μL;

[0072] The detection wavelength is 210nm;

[0073] The chromatographic separation results are shown in the following table:

[0074] Sample name RT(min) R Acetic acid 6.155 / 1.3-Acetonedicarboxylic acid 7.902 7.60 Acetoacetic acid 9.385 8.21 Citric Acid 10.414 4.46 Cis-aconitic acid 16.023 14.02 trans-aconitic acid 20.027 8.63 Itaconic acid 29.800 15.45

[0075] It can be seen from the results in the above table that the chromatographic conditions studied in this embodiment can completely separate citric acid and its six organic acid impurities with good separation, meeting the requirements of the Chinese Pharmacopoeia; the chromatographic separation spectrum results are shown in Figure 4 .

[0076] Example 5

[0077] A high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, the steps of which are as follows: Weigh 40 mg of acetic acid standard, 20 mg of 1.3-acetone dicarboxylic acid standard, 20 mg of acetoacetic acid, 20 mg of cis-aconitic acid standard, 20 mg of trans-aconitic acid standard, and 20 mg of itaconic acid standard, accurately weigh in a 100 mL volumetric flask, dissolve to scale with water, mix well, and use as an impurity reserve solution; then accurately transfer 5.0 mL of the solution to a 100 mL volumetric flask, dilute to scale with water, mix well, and use as an impurity mother liquor. Weigh 70 mg of citric acid standard, accurately weigh in a 50 mL volumetric flask, first add a small amount of water to dissolve, then add 5.0 mL of impurity mother liquor, dilute to scale with water, mix well, and use as a separation solution. That is, a high performance liquid chromatograph is used with octadecyl bonded silica gel as a filler. AQ-C18 5μm×4.6mm×250mm is used as the chromatographic column, and the separation is performed under the following separation conditions:

[0078] Mobile phase: phosphoric acid aqueous solution, pH = 2.00;

[0079] The mobile phase flow rate was 1.0 mL / min;

[0080] The column temperature was 30°C;

[0081] The injection volume was 20 μL;

[0082] The detection wavelength is 210nm;

[0083] The chromatographic separation results are shown in the following table:

[0084] Sample name RT(min) R Acetic acid 4.641 / 1.3-Acetonedicarboxylic acid 5.193 3.35 Acetoacetic acid 5.525 2.51 Citric Acid 6.105 3.75 Cis-aconitic acid 8.200 8.75 trans-aconitic acid 10.387 8.05 Itaconic acid 13.451 9.02

[0085] It can be seen from the results in the above table that the chromatographic conditions studied in this embodiment can completely separate citric acid and its six organic acid impurities with good separation, meeting the requirements of the Chinese Pharmacopoeia; the chromatographic separation spectrum results are shown in Figure 5 .

[0086] Example 6

[0087] A high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, the steps of which are as follows: Weigh 40 mg of acetic acid standard, 20 mg of 1.3-acetone dicarboxylic acid standard, 20 mg of acetoacetic acid, 20 mg of cis-aconitic acid standard, 20 mg of trans-aconitic acid standard, and 20 mg of itaconic acid standard, accurately weigh in a 100 mL volumetric flask, dissolve to scale with water, mix well, and use as an impurity reserve solution; then accurately transfer 5.0 mL of the solution to a 100 mL volumetric flask, dilute to scale with water, mix well, and use as an impurity mother liquor. Weigh 70 mg of citric acid standard, accurately weigh in a 50 mL volumetric flask, first add a small amount of water to dissolve, then add 5.0 mL of impurity mother liquor, dilute to scale with water, mix well, and use as a separation solution. That is, a high performance liquid chromatograph is used with octadecyl bonded silica gel as a filler. AQ-C18 5μm×4.6mm×250mm is used as the chromatographic column, and the separation is performed under the following separation conditions:

[0088] Mobile phase: 40mmol / L ammonium dihydrogen phosphate aqueous solution, pH = 2.00;

[0089] The mobile phase flow rate was 1.0 mL / min;

[0090] The column temperature was 30°C;

[0091] The injection volume was 20 μL;

[0092] The detection wavelength is 210nm;

[0093] The chromatographic separation results are shown in the following table:

[0094] Sample name RT(min) R Acetic acid 4.462 / 1.3-Acetonedicarboxylic acid 5.153 4.53 Acetoacetic acid 5.777 4.82 Citric Acid 6.736 4.73 Cis-aconitic acid 9.236 8.79 trans-aconitic acid 11.529 7.61 Itaconic acid 15.021 9.31

[0095] It can be seen from the results in the above table that the chromatographic conditions studied in this embodiment can completely separate citric acid and its six organic acid impurities with good separation, meeting the requirements of the Chinese Pharmacopoeia; the chromatographic separation spectrum results are shown in Figure 6 .

[0096] Example 7

[0097] The calibration of the citric acid peak position in a method for separating ferric citrate and six organic acids thereof is as follows: weigh 70 mg of citric acid standard, accurately weigh it in a 50 mL volumetric flask, dissolve it in water and dilute it to the scale, mix it, and use it as a reference solution. According to the chromatographic conditions: Hydro-RP 80A 4μm×4.6mm×250mm with octadecyl bonded silica as the filler is used as the chromatographic column, and the separation is performed under the following separation conditions:

[0098] Mobile phase: phosphoric acid aqueous solution, pH = 2.00;

[0099] The mobile phase flow rate was 1.0 mL / min;

[0100] The column temperature was 30°C;

[0101] The injection volume was 20 μL;

[0102] The detection wavelength is 210nm;

[0103] The chromatographic separation results are as follows Figure 7 As shown, from Figure 7 It can be seen that the retention time of citric acid is 10.414 min. Compared with Example 1, since Example 7 uses the same liquid phase method as Example 1, it is indicated that the peak at about 10 min in Example 1 and other examples is citric acid.

[0104] Example 8

[0105] The calibration of the citric acid peak position in a method for separating ferric citrate and six organic acids is as follows: weigh 70 mg of citric acid standard, accurately weigh it in a 50 mL volumetric flask, dissolve it in water and dilute it to the scale, mix it, and use it as a reference solution. According to the chromatographic conditions: octadecyl bonded silica gel is used as the filler AQ-C18 5μm×4.6mm×250mm is used as the chromatographic column, and the separation is performed under the following separation conditions:

[0106] Mobile phase: phosphoric acid aqueous solution, pH = 2.00;

[0107] The mobile phase flow rate was 1.0 mL / min;

[0108] The column temperature was 30°C;

[0109] The injection volume was 20 μL;

[0110] The detection wavelength is 210nm;

[0111] The chromatographic separation results are as follows Figure 8 As shown, from Figure 8 It can be seen that the retention time of citric acid is 6.110 min. Compared with Example 5, since Example 8 and Example 5 use the same liquid phase method, it is indicated that the peak at around 6 min in Example 5 and other examples is citric acid.

[0112] In summary, the HPLC separation and analysis method of ferric citrate and six organic acids of the present invention can effectively separate citric acid from the other six organic acid impurities, and the separation degree of the Hydro-RP 80A 4μm×4.6mm×250mm column can be as high as 4.46-15.45; The resolution of the AQ-C18 5μm×4.6mm×250mm column can be as high as 2.51-9.31, which meets the requirements of the Chinese Pharmacopoeia.

Claims

1. A high performance liquid chromatography separation and analysis method for ferric citrate and six organic acids thereof, characterized in that: The organic acid is acetic acid, 1,3-acetone dicarboxylic acid, acetoacetic acid, citric acid, cis-aconitic acid, trans-aconitic acid, and itaconic acid; A high performance liquid chromatograph was used, and an Ultimate® AQ-C18 5μm×4.6mm×250mm column or a Hydro-RP 80A 4μm×4.6mm×250mm column with octadecyl bonded silica as a filler was selected as a chromatographic column, and an aqueous phosphoric acid solution and a phosphate buffer solution were used as mobile phases. The mobile phase was an aqueous phosphoric acid solution, and the pH was adjusted to 2.00 with orthophosphoric acid or / and a 40mmol / L diammonium phosphate buffer solution, and the pH was adjusted to 2.00 with orthophosphoric acid. The following chromatographic separation conditions were used for separation: The mobile phase flow rate is 0.80-1.20 mL / min; The column temperature was 30°C; The injection volume was 20 μL; The detection wavelength is 210-240nm; The elution method is isocratic elution, and the isocratic elution conditions are as follows: 。 2. The high performance liquid chromatography separation and analysis method of ferric citrate and six organic acids thereof as claimed in claim 1, characterized in that The flow rate of the mobile phase is 1.0 mL / min, the temperature of the chromatographic column is 30° C., and the detection wavelength is 210 nm.

Images