Dexrazoxane analytical method

Abstract

A high performance liquid chromatography method used for dexrazoxane-related substances is provided, and in the method, a low-density bonding reversed-phase C18 chromatographic column resistant to pure water is employed; a gradient elution is carried out with mobile phase A and mobile phase B as eluents, the mobile phase A being a buffer, and the mobile phase B being an organic solvent; the volume percent of mobile phase A in eluents in a first stage of the gradient elution is not lower than 90%, and the duration of the first stage of the gradient elution ranges from 15˜30 minutes. By means of the analytical method, dexrazoxane is effectively separated from main impurities, and the qualities of the active pharmaceutical ingredients of dexrazoxane and the preparations thereof could be better controlled.

Claims

1. A high performance liquid chromatography method used for dexrazoxane-related substances, comprising: performing a gradient elution via a low-density bonding reversed-phase chromatographic column resistant to pure water, where the gradient elution is carried out with a mobile phase A and a mobile phase B as eluents; wherein the mobile phase A is a buffer; the mobile phase B is an organic solvent; and the gradient elution comprises at least a first stage that is 15-30 minutes in duration in which the volume of the mobile phase A that is introduced into the low-density bonding reversed-phase C18 chromatographic column is not lower than is such that the mobile phase A represents 90% or more of the total volume of all eluents of the mobile phase being introduced into the low-density bonding reversed-phase C18 chromatographic column.

2. The method of claim 1, wherein the low-density bonding reversed-phase C18 chromatographic column resistant to pure water is Waters Atlantis T3 or Waters ACQUITY HSS T3.

3. The method of claim 2, wherein the low-density bonding reversed-phase C18 chromatographic column resistant to pure water is Waters Atlantis T3.

4. The method of claim 1, wherein the buffer is selected from a buffered salt solution of 1-50 mmol/L at a pH of 1-6, of potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate or ammonium dihydrogen phosphate; and the organic solvent is selected from methanol, acetonitrile or isopropanol.

5. The method of claim 4, wherein the buffer is a potassium dihydrogen phosphate solution of 5-15 mmol/L at a pH of 3.5-5.5, the organic solvent is methanol or acetonitrile.

6. The method of claim 4, wherein the buffer is a potassium dihydrogen phosphate solution of 8-12 mmol/L at a pH of 4.5-5, the organic solvent is methanol.

7. The method of claim 1, wherein the mobile phase A represents 95% or more of the total volume of all the eluents of the mobile phase introduced into the low-density bonding reversed-phase C18 chromatographic column in the first stage of the gradient elution, and the duration of the first stage of the gradient elution ranges from 15-20 minutes.

8. The method of claim 1, wherein the gradient elution further comprises a second, a third and a fourth stage; the only eluent introduced into the low-density bonding reversed-phase C18 chromatographic column during the first stage of the gradient elution is the mobile phase A, the first stage ending 15 minutes after it begins; during the second stage, which lasts for a duration of 30 minutes and immediately follows the first stage, the volume percent of the mobile phase A introduced into the low-density bonding reversed-phase C18 chromatographic column is adjusted from 100% to a predetermined volume percent of n %, where 10≤n≤90, and, at the same time, the volume percent of the mobile phase B introduced into the low-density bonding reversed-phase C18 chromatographic column in the second stage of the gradient elution is adjusted from 0% to a volume percent of (100−n) %, wherein during the entire second stage the volume percentages of the mobile phase A and the mobile phase B are adjusted in a linear manner such that the predetermined volume percent of n % for the mobile phase A is achieved at the 30 minute mark of the second stage; during the third stage, which lasts for a duration of 5 minutes and immediately follows the second stage, the volume percent of the mobile phase A introduced into the low-density bonding reversed-phase C18 chromatographic column is maintained at the predetermined volume percent of n % that was achieved at the end of the second stage, and the volume percent of the mobile phase B introduced into the low-density bonding reversed-phase C18 chromatographic column is maintained at the volume percent of (100−n) % that was achieved at the end of the second stage; and during the fourth stage, which lasts for a duration of 10 minutes and immediately follows the third stage, in the initial 0.01 minute of the fourth stage the volume percent of the mobile phase A introduced into the low-density bonding reversed-phase C18 chromatographic column is increased from the predetermined volume percent of n % that was maintained for 5 minutes in the third stage to 100%, and at the same time, the volume percent of the mobile phase B introduced into the low-density bonding reversed-phase C18 chromatographic column is reduced to 0%, and then for the remaining 9.99 minutes of the fourth stage the only eluent introduced into the low-density bonding reversed-phase C18 chromatographic column is the mobile phase A.

9. The method of claim 8, wherein 50≤n≤80.

10. The method of claim 8, wherein 60≤n≤80.

11. The method of claim 1, wherein the flow rate of the mobile phase is 0.6-1.5 mL/min, and/or the temperature of the chromatographic column is 5-20° C., and/or the ultraviolet detector is employed as the detector, with a detection wavelength of 200-220 nm.

12. The method of claim 11, wherein the flow rate of the mobile phase is 0.8-1.2 mL/min; the temperature of the chromatographic column is 10-20° C.; the detection wavelength is 203-213 nm.

13. The method of claim 1, further comprising: preparing a test solution from an amount of dexrazoxane or dexrazoxane-containing related preparations and a diluent, the test solution having a concentration of 0.5-2 mg dexrazoxane per 1 mL; preparing a control solution from the test solution by diluting an aliquot of the test solution to a volume of 100 times that of the aliquot with a hydrochloric acid solution; preparing a system suitability test solution from a hydrochloric acid solution and an amount of dexrazoxane or dexrazoxane-containing related preparations, an impurity I control, an impurity II control, and an impurity III control the system suitability test solution containing dexrazoxane at 0.5-2 mg, impurity I at 5-20 μg, impurity II at 5-20 μg, and impurity III at 5-20 μg per 1 mL; injecting 10-20 μl of the system suitability test solution into a liquid chromatograph that comprises the low-density bonding reversed-phase C18 chromatographic column, injecting 10-20 μl of the control solution into the liquid chromatograph, and injecting 10-20 μl of test solution into the liquid chromatograph and recording the chromatogram; wherein the diluent is a hydrochloric acid solution, the chemical structures of impurity I, impurity II, impurity III are shown as below: ##STR00003##

14. The method of claim 13, wherein the test solution has a concentration of 0.8-1.1 mg of the dexrazoxane per 1 mL; the control solution contains the dexrazoxane at 0.8-1.1 mg, the impurity I at 8-12 μg, the impurity II at 8-12 μg, and the impurity III at 8-12 μg per 1 mL; 10 μl of the system suitability test solution 10 μl is injected, into the liquid chromatograph; 10 μl of the control solution 10 μl is injected into the liquid chromatograph; and 10 μl of the test solution is injected into the liquid chromatograph.

15. The method of claim 1, further comprising detecting the presence of dexrazoxane and/or one or more impurities in a sample or dexrazoxane-containing preparations.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the chromatogram under the conditions of the control example 1;

(2) FIG. 2 is the chromatogram under the conditions of the control example 2;

(3) FIG. 3 is the chromatogram under the conditions of the control example 3;

(4) FIG. 4 is the chromatogram under the conditions of the control example 4;

(5) FIG. 5 is the chromatogram of the system suitability test under the conditions of Embodiment 1;

(6) FIG. 6 is the chromatogram of the test solution under the conditions of Embodiment 1.

DETAILED DESCRIPTION

(7) The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to further illustrate the present invention, rather than imposing any limits on the scope of the present invention. Dexrazoxane (the active pharmaceutical ingredients), lyophilized preparations of dexrazoxane, impurity I control, impurity II control, impurity III control used in the detailed description of the present invention are all from Jiangsu Aosaikang Pharmaceutical Co., Ltd.; the instrument being used is Agilent 1290 high-performance liquid chromatograph, including G4220B 1290 Bin Pump VL pump, G4212B 1260DAD ultraviolet detector and Thermo Chromeleon 7.2 chromatographic working station. The percentage contents described in the course of elution in the following control examples and embodiments, refer to the volume percent contents.

Control Example 1

(8) 1. Chromatographic Conditions

(9) Chromatographic column: Inertsil ODS-SP chromatographic column (150′4.6 mm, 5 μm)

(10) Mobile phase: methanol-0.01 mol/L solution of potassium dihydrogen phosphate (15:85)

(11) Detection wavelength: 208 nm

(12) Flow rate: 1.0 mL/min

(13) Column temperature: 30° C.

(14) 2. Experimental Steps

(15) Taking dexrazoxane 10 mg, precisely determined, into which was added 1.0 mL NaOH solution at 0.1 mol/L, left at room temperature for 10 minutes, 0.1 mol/L HCl solution was added to neutralize, and diluting it with a solution of potassium dihydrogen phosphate at 0.01 mol/L to make a solution containing about 1 mg dexrazoxane per 1 mL, which was used as the test solution. [Note: Into the control examples 1-4 was added 1.0 mL NaOH solution at 0.1 mol/L to achieve the effect of alkaline destruction, significantly enhancing the contents of impurity I, impurity II, impurity III in the test solution.

(16) Following the high-performance liquid chromatography (Chinese Pharmacopoeia, Edition 2005, Part 2, Appendix VD), the determination was performed under the above conditions. Taking 10 μl test solution, injecting it into the liquid chromatograph, adjusting the detection sensitivity, making the peak height of the chromatographic peak of the main ingredient to be about 20-30% of the full scale, recording the chromatogram, with the results seen in the accompanying FIG. 1 and Table 1.

(17) As can be seen from the detection results, the chromatographic peaks of impurities III, II, I were overlapped under this condition, the retention time of dexrazoxane was about 4.4 min, and the resolution between impurities was not up to standard. It can be detected by this method that the number of impurities in the sample was not more than 5.

(18) TABLE-US-00003 TABLE 1 Results of the test on the separation between dexrazoxane and related substances under the condition of the control example 1 Impurity III Impurity II Impurity I Dexrazoxane Number of Number of Number of Number of Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Time lution Plates Time lution Plates Time lution Plates Time lution Plates Control Maybe — — 1.786 1.00 3198 1.926 13.00 2445 4.446 14.74 5826 Example overlapped 1 with impurity II Note: “—” indicted not detected.

Control Example 2

(19) 1. Chromatographic Conditions

(20) Chromatographic column: Dikma Spursil ODS chromatographic column (250*4.6 mm, 5 μm)

(21) Mobile phase: A is a solution of sodium dihydrogen phosphate at 0.01 mol/L

(22) B is methanol

(23) Detection wavelength: 208 nm

(24) Flow rate: 1.0 mL/min

(25) Column temperature: 30° C.

(26) Gradient elution conditions:

(27) TABLE-US-00004 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 96 4 15 96 4 25 90 10 40 85 15 50 85 15 50.01 96 4 60 96 4

(28) 2. Experimental Steps

(29) Taking dexrazoxane 10 mg, precisely determined, into which was added 1.0 mL NaOH solution at 0.1 mol/L, left at room temperature for 10 minutes, 0.1 mol/L HCl solution was added to neutralize, and diluting it with a solution of potassium dihydrogen phosphate at 0.01 mol/L to make a solution containing about 1 mg dexrazoxane per 1 mL, which was used as the test solution.

(30) Following the high-performance liquid chromatography (Chinese Pharmacopoeia, Edition 2015, Part 4, Appendix 0512), the determination was performed under the above conditions. Taking 10 μl test solution, injecting it into the liquid chromatograph, adjusting the detection sensitivity, making the peak height of the chromatographic peak of the main ingredient to be about 20-30% of the full scale, recording the chromatogram, with the results seen in the accompanying FIG. 2 and Table 2.

(31) As can be seen from the detection results, the chromatographic peaks of impurities III, II, I were overlapped under this condition, the chromatographic peak of impurity III was overlapped with the peak of the blank solvent, the retention time of dexrazoxane was about 16.2 min, and the resolution between impurities was not up to standard.

(32) TABLE-US-00005 TABLE 2 Results of the test on the separation between dexrazoxane and related substances under the condition of the control example 2 Impurity III Impurity II Impurity I Dexrazoxane Number of Number of Number of Number of Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Time lution Plates Time lution Plates Time lution Plates time lution Plates Control 2.905 1.28 6764 3.139 1.22 3168 3.472 24.41 1804 16.212 22.90 8117 Example (overlapped 2 with the peak of the blank solvent)

Control Example 3

(33) 1. Chromatographic Conditions

(34) Chromatographic column: Waters XBridge Hilic chromatographic column (250*4.6 mm, 5 μm)

(35) Mobile phase: A is a solution of ammonium acetate at 0.01 mol/L

(36) B is acetonitrile

(37) Detection wavelength: 208 nm

(38) Flow rate: 1.0 mL/min

(39) Column temperature: 30° C.

(40) Gradient elution conditions:

(41) TABLE-US-00006 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 5 95 30 50 50 50 50 50 50.01 5 95 60 5 95

(42) 2. Experimental Steps

(43) Taking dexrazoxane 10 mg, precisely determined, into which was added 1.0 mL NaOH solution at 0.1 mol/L, left at room temperature for 10 minutes, 0.1 mol/L HCl solution was added to neutralize, and diluting it with a solution of ammonium acetate at 0.01 mol/L to make a solution containing about 1 mg dexrazoxane per 1 mL, which was used as the test solution.

(44) Following the high-performance liquid chromatography (Chinese Pharmacopoeia, Edition 2015, Part 4, Appendix 0512), the determination was performed under the above conditions. Taking 10 μl test solution, injecting it into the liquid chromatograph, adjusting the detection sensitivity, making the peak height of the chromatographic peak of the main ingredient to be about 20-30% of the full scale, recording the chromatogram, with the results seen in the accompanying FIG. 3.

(45) It was shown from the detection results that the retention time of the main peak was very short and the main peak was splitted, the peak shape of the impurities was not desirable and the baseline was not smooth. This method can't meet the criteria in Chinese Pharmacopoeia.

Control Example 4

(46) 1. Chromatographic Conditions

(47) Chromatographic column: YMC-Pack ODS-AQ chromatographic column (1504.6 mm, 5 μm)

(48) Mobile phase: A is a solution of potassium dihydrogen phosphate at 0.01 mol/L

(49) B is methanol

(50) Detection wavelength: 208 nm

(51) Flow rate: 1.0 mL/min

(52) Column temperature: 20° C.

(53) Gradient elution conditions:

(54) TABLE-US-00007 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 10 96 4 20 90 10 30 90 10 40 80 20 50 80 20 50.01 100 0 60 100 0

(55) 2. Experimental Steps

(56) Taking dexrazoxane 10 mg, precisely determined, into which was added 1.0 mL NaOH solution at 0.1 mol/L, left at room temperature for 10 minutes, 0.1 mol/L HCl solution was added to neutralize, and diluting it with a solution of potassium dihydrogen phosphate at 0.01 mol/L to make a solution containing about 1 mg dexrazoxane per 1 mL, which was used as the test solution.

(57) Following the high-performance liquid chromatography (Chinese Pharmacopoeia, Edition 2015, Part 4, Appendix 0512), the determination was performed under the above conditions. Taking 10 μl test solution, injecting it into the liquid chromatograph, adjusting the detection sensitivity, making the peak height of the chromatographic peak of the main ingredient to be about 20-30% of the full scale, recording the chromatogram, with the results seen in the accompanying FIG. 4 and Table 3.

(58) According to detection results, the chromatographic peak of impurity III was overlapped with the peak of the blank solvent, and this method can't meet the criteria in Chinese Pharmacopoeia.

(59) TABLE-US-00008 TABLE 3 Results of the test on the separation between dexrazoxane and related substances under the condition of the control example 4 Impurity III Impurity II Impurity I Dexrazoxane Number of Number of Number of Number of Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Time lution Plates Time lution Plates Time lution Plates Time lution Plates Control — — — 3.519 5.47 1775 5.272 47.26 4681 21.059 — — Example (overlapped 4 with the peak of the blank solvent) Note: “—” indicted not detected or no data obtained.

Embodiment 1

(60) 1. Chromatographic Conditions

(61) Chromatographic column: Waters Atlantis T3 chromatographic column (250*4.6 mm, 5 μm)

(62) Mobile phase: A is a solution of KH.sub.2PO.sub.4 at 10 mmol/L (pH4.5˜5)

(63) B is methanol

(64) Detection wavelength: 208 nm

(65) Flow rate: 1.0 mL/min

(66) Column temperature: 15° C.

(67) Gradient elution conditions:

(68) TABLE-US-00009 Mobile phase B Time (minutes) Mobile phase A (%) (%) First stage 0 100 0 15 100 0 45 70 30 50 70 30 50.01 100 0 60 100 0

(69) 2. Experimental Steps

(70) Taking the lyophilized preparations of dexrazoxane, precisely determined, into which was added a hydrochloric acid solution at 0.1 mol/L, and quantitatively diluted to a solution containing about 1 mg dexrazoxane per 1 mL, which was used as the test solution. 1 mL test solution was precisely measured and placed into a 100 mL volumetric flask, diluted to the scale with 0.1 mol/L hydrochloric acid, shaked well, and used as the control solution. Additionally, taking proper amounts of the lyophilized preparations of dexrazoxane, impurity I, impurity II, impurity III controls, into which was added a hydrochloric acid solution at 0.1 mol/L, and diluted to a solution containing about 1 mg dexrazoxane and impurities each 10 μg per 1 mL, which was used as the system suitability test solution.

(71) Following the high-performance liquid chromatography (Chinese Pharmacopoeia, Edition 2015, Part 4, Appendix 0512), the determination was performed under the above conditions. Taking 10 μl of the system suitability test solution, injecting it into the liquid chromatograph, recording the chromatogram, with the results seen in the accompanying FIG. 5 and Table 4.

(72) As can be seen from the detection results, impurity III, impurity II, impurity I and dexrazoxane are separated out successively, the retention time was 4.7 min, 7.2 min, 12.7 min, 34.9 min, respectively, and the resolution between impurities as well as between impurities and main peaks were all greater than 5, meeting the criteria in Chinese Pharmacopoeia. Compared with the control examples, the numbers of theoretical plates of impurities and main peaks in the method of the present invention were higher, and more impurities could be detected in the sample, with a general number of 15˜25.

(73) TABLE-US-00010 TABLE 4 Results of the system suitability test on the separation between dexrazoxane and related substances under the condition of Embodiment 1 Impurity III Impurity II Impurity I Dexrazoxane Number of Number of Number of Number of Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Time lution Plates Time lution Plates Time luton Plates Time lution Plates Embodiment 1 4.691 11.70 9960 7.178 6.84 14529 12.751 44.04 16487 34.918 26.32 253171

(74) Taking the control solution 10 μl, injecting it into the liquid chromatograph, adjusting the detection sensitivity, making the peak height of the chromatographic peak of the main ingredient to be about 10% of the full scale. Precisely measuring the test solution and the control solution 10 μl each, respectively being injected into the liquid chromatograph, and recording the chromatogram, wherein the chromatogram of the test solution was shown in the accompanying FIG. 6. Detection results show that the impurity I was not detected, the resolutions between impurity II and I as well as between the impurities and the main peaks were both well, thus meeting the criteria in Chinese Pharmacopoeia.

Embodiments 2˜9

(75) 1. Chromatographic Conditions

(76) The conditions of the chromatographic column and the gradient elution employed in the chromatographic conditions in Embodiments 2˜9 were the same as those in Embodiment 1, and other chromatographic conditions were shown as below:

(77) TABLE-US-00011 Detection Column Mobile Phase Wavelength Flow Rate Temperature Embodiment 2 A: KH.sub.2PO.sub.4 solution (8 mmol/L, pH4.5~5) 208 nm 1.0 mL/min 15 B: Methanol Embodiment 3 A: KH.sub.2PO.sub.4 solution (12 mmol/L, pH 4.5~5) 208 nm 1.0 mL/min 15 B: Methanol Embodiment 4 A: KH.sub.2PO.sub.4 solution (10 mmol/L, pH 4.5~5) 203 nm 1.0 mL/min 15 B: Methanol Embodiment 5 A: KH.sub.2PO.sub.4 solution (10 mmol/L, pH 4.5~5) 213 nm 1.0 mL/min 15 B: Methanol Embodiment 6 A: KH.sub.2PO.sub.4 solution (10 mmol/L, pH 4.5~5) 208 nm 0.8 mL/min 15 B: Methanol Embodiment 7 A: KH.sub.2PO.sub.4 solution (10 mmol/L, pH 4.5~5) 208 nm 1.2 mL/min 15 B: Methanol Embodiment 8 A: KH.sub.2PO.sub.4 solution (10 mmol/L, pH 4.5~5) 208 nm 1.0 mL/min 10 B: Methanol Embodiment 9 A: KH.sub.2PO.sub.4 solution (10 mmol/L, pH 4.5~5) 208 nm 1.0 mL/min 20 B: Methanol

(78) 2. Experimental Steps

(79) Taking the lyophilized preparations of dexrazoxane, the liquid chromatography was performed according to the experimental steps described in Embodiment 1 under the chromatographic conditions of the above embodiments 2˜9. The results of the test solution detection were shown in Table 5:

(80) TABLE-US-00012 TABLE 5 Results of the system suitability test on the separation between dexrazoxane and related substances under the conditions of Embodiment 2~9 Impurity III Impurity II Impurity I Dexrazoxane Number of Number of Number of Number of Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Retention Reso- Theoretical Time lution Plates Time lution Plates Time lution Plates Time lution Plates Embodiment 2 — — — 7.190 3.91 9091 12.817  2.10 9887 34.904 — 142908 Embodiment 3 — — — 7.182 5.14 8431 12.782 17.43 9166 34.902 14.94 133939 Embodiment 4 — — — 7.183 4.98 9708 12.783 32.84 10589 34.910 — 112941 Embodiment 5 — — — 7.177 5.03 9658 12.777  8.86 10583 34.904 — 183775 Embodiment 6 — — — 8.946 5.57 11717 15.920 18.06 12748 37.053 20.08 143025 Embodiment 7 — — — 5.989 4.78 8936 10.663 — 9799 33.316 16.21 155680 Embodiment 8 — — — 7.725 5.78 9932 14.225 27.09 10917 35.625 17.24 153198 Embodiment 9 — — — 6.717 4.67 10987 11.584 — 12092 34.218 21.73 157645 Note: “—” indicted not detected or no data obtained.

(81) It was shown from the results that the resolutions between the main peak and the impurity peak and the number of theoretical plates both met the criteria when there were slight changes in the concentration of the buffer, the wavelength, the flow rate and the column temperature, and the durability for the method of the present invention would be good.

(82) In addition, the method of the present invention was validated in the terms of methodology through various tests on the specificity, the quantitation limit and the detection limit, the repeatability, the accuracy, or the like, and the results indicated that the method of the present invention had a good specificity, a high sensitivity, and were well in both the repeatability and the accuracy, capable of meeting the need of the analytical detection on the active pharmaceutical ingredients of dexrazoxane and their preparations.