Method for detecting nitro10-nitrosofolinic acid in calcium folinate injection by adopting liquid chromatography-mass spectrometry

Abstract

The invention discloses a method for detecting nitro10-nitrosofolinic acid in calcium folinate injection by liquid chromatography. According to the method, a phenyl column is used as a chromatographic column, a 0.1% formic acid aqueous solution is used as a mobile phase A, methanol is used as a mobile phase B, a gradient elution mode is adopted, and an ion source is ESI (+). The method has the advantages of good specificity and stable separation effect, is suitable for determining nitrogen-10-nitrosofolinate in the calcium folinate injection, and has the sensitivity of 0.09 ppm.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting N10-nitrosomethylfolate in calcium folinate injection by liquid chromatography-mass spectrometry. Background Art

[0002] Calcium folinate is the calcium salt of the formyl derivative of calcium tetrahydrofolate. It is an anti-anemic drug that can counteract the toxicity of acid antagonists (such as methotrexate, pyrimethamine, and trimethoprim) and can treat megaloblastic anemia caused by folic acid deficiency, promoting the differentiation, maturation, and release of bone marrow hematopoietic cells. The structure of this product contains a secondary amine structure. According to the "Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities" guidelines issued by the FDA, in its injection, it may react with trace amounts of nitrite ions that may be present in the excipients to form the drug matrix nitrosamine impurity: N10-nitrosomethylfolate. Therefore, it is necessary to detect and control the content of N10-nitrosomethylfolate in calcium folinate injection. By consulting the literature and data, there is no report on the determination method of N10-nitrosomethylfolate.

[0003]

[0004] Since N10-nitrosomethylfolate has a high structural similarity to calcium folinate, it is difficult to improve the separation degree. After a large number of chromatographic condition screenings and methodological studies by the inventor of the present invention, it was finally found that using a phenyl column can improve the separation degree. This method has good specificity, stable separation effect, is applicable to the determination of N10-nitrosomethylfolate in calcium folinate injection, and the sensitivity can reach 0.09 ppm. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting N10-nitrosomethylfolate in calcium folinate injection by liquid chromatography-mass spectrometry, which has high specificity, stable separation effect, and high sensitivity.

[0006] To achieve the purpose of the present invention, the following implementation schemes are provided: In one implementation scheme, the method for N10-nitrosomethylfolate in the calcium folinate injection of the present invention is characterized in that: the method includes: using a phenyl column as the chromatographic column, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is methanol, adopting gradient elution mode, and the ion source is ESI(+).

[0007] Preferably, for the method of the present invention, the phenyl column is Phenomenex Luna 3μm Phenyl-hexyl 100A, 150×4.6mm, 3μm.

[0008] Preferably, in the method of the present invention, the initial volume ratio of mobile phase A to mobile phase B is 70:30.

[0009] Preferably, in the method of the present invention, gradient elution is adopted, and the program is as follows:

[0010] Furthermore, the method of the present invention further includes: the column temperature is 30°C, the flow rate is 0.5 ml / min, and the sample volume is 10 μl.

[0011] Furthermore, for the method of the present invention, the mass spectrometry parameters are as follows:

[0012] Preferably, in the method of the present invention, the content of nitrogen 10-nitrosoglutamate in the test solution is calculated by the external standard method of reference substance. Description of the Drawings

[0013] Figure 1 It is the chromatogram of the blank solvent in Example 1; Figure 2 It is the chromatogram of the control solution in Example 1; Figure 3 It is the chromatogram of the test solution in Example 1; Figure 4 It is the chromatogram of the control solution in Example 2; Figure 5 It is the chromatogram of the test solution in Example 2; Figure 6 It is the chromatogram of the spiked test solution in Example 2; Figure 7 It is the chromatogram of the LOD solution in Example 3; Figure 8 It is the chromatogram of the LOG solution in Example 3; Figure 9 It is the chromatogram of the reference substance solution of Waters XBridge C18 column in Example 4; Figure 10 It is the chromatogram of the test solution of Waters XBridge C18 column in Example 4; Figure 11 It is the chromatogram of the 100% limit level spiked test solution of Waters XBridge C18 column in Example 4; Figure 12 It is the chromatogram of the reference substance solution of ZORBAX SB-C18 column in Example 4; Figure 13Chromatogram of the test solution for the ZORBAX SB-C18 chromatographic column in Example 4; Figure 14 Chromatogram of the reference solution for the ACE Excel C18-AR chromatographic column in Example 4; Figure 15 Chromatogram of the test solution for the ACE Excel C18-AR chromatographic column in Example 4; Figure 16 Chromatogram of the spiked test solution at the 100% limit level for the ACE Excel C18-AR chromatographic column in Example 4. Detailed implementation mode

[0014] The following examples illustrate the technical solutions of the present invention in detail to help understand the essence of the present invention, but do not limit the scope of the present invention in any way.

[0015] Example 1 The chromatographic conditions are as follows:

[0016] Quantification method: Calculate the content of 10-nitrosoglutamic acid in the test solution by the external standard method with reference substance.

[0017] Detection process: Solution preparation (1) Mobile phase A: 0.1% formic acid in water: Pipette 500 ml of ultrapure water, add 0.5 ml of formic acid, mix well and sonicate for 15 min to obtain.

[0018] (2) Mobile phase B: Methanol (3) Solvent: Pure water (4) Reference solution: Weigh an appropriate amount of 10-nitrosoglutamic acid reference substance accurately, quantitatively dilute it with diluent DMSO to prepare a solution containing about 0.1 mg of 10-nitrosoglutamic acid per 1 ml, then take an appropriate amount of this solution and quantitatively dilute it with pure water to prepare a solution containing about 10 ng of 10-nitrosoglutamic acid per 1 ml.

[0019] (5) Test solution: Calcium folinate injection (10 mg / ml).

[0020] Detection: Inject blank solvent water, reference solution and test solution into the gas chromatography-mass spectrometry instrument respectively, and record the chromatogram.

[0021] The results are shown in Figure 1 , Figure 2 and Figure 3 , where Figure 1 is the blank solvent, Figure 2 is the reference solution, Figure 3 is the test solution.

[0022] The results showed that there was no interference in the blank solvent and the test sample, and the specificity was good.

[0023] Quantification method: The impurity content was calculated by the external standard method (the target impurity was a double peak, and the integration was combined). The formula was as follows:

[0024] Test results of the sample:

[0025] Example 2 Accuracy test The chromatographic conditions were the same as those in Example 1 Solution preparation: The test solution and the reference solution were the same as those in Example 1. Spiked stock solution: Take an appropriate amount of the N10-nitrosofolic acid reference substance, weigh it accurately, quantitatively dilute it with DMSO to prepare a solution containing about 0.1 mg of N10-nitrosofolic acid per 1 ml, and then take an appropriate amount of this solution and quantitatively dilute it with pure water to prepare a solution containing about 0.3 μg of N10-nitrosofolic acid per 1 ml Spiked test solution: (Preparation method) Accurately pipette 985 μL of the test solution, add 15 μL of the spiked stock solution, and mix well to obtain.

[0026] Test: Inject the test solution, the reference solution and the spiked test solution into the chromatograph-mass spectrometer and record the chromatograms.

[0027] The results are shown in Figures 4 - 6 , where Figure 4 is the reference solution, Figure 5 is the test solution, Figure 6 is the spiked test solution.

[0028] The results showed that: after spiking the test solution, the recovery was good and the method accuracy was good.

[0029] Example 3 Sensitivity test The chromatographic conditions were the same as those in Example 1 Solution preparation: Reference solution: The same as that in Example 1.

[0030] LOD solution preparation: Accurately pipette 0.5 ml of the reference solution and place it in a 10 ml volumetric flask, dilute it to the mark with the diluent, and shake well to obtain.

[0031] LOQ solution: Accurately pipette 1 ml of the reference solution and place it in a 10 ml volumetric flask, dilute it to the mark with the diluent, and shake well to obtain.

[0032] Test: Inject LOD solutions and LOQ solutions at various concentrations into the gas chromatography-mass spectrometry instrument and record the chromatograms.

[0033] The results are shown in Figures 7 - 8 , where Figure 7 is the limit of the LOD solution (0.05 ppm), Figure 8 is the limit of the LOQ solution (0.09 ppm).

[0034] The results show that the detection limit of this method can reach 0.05 ppm, the quantitation limit is 0.09 ppm, and the method has high sensitivity.

[0035] Example 4 Condition Parameter Screening 1. Chromatographic Column Change Replace the chromatographic column Luna phenyl-hexyl, 150*4.6 mm, 3 μm in Example 1 with Waters XBridge C18, 150*4.5 mm, 3.5 μm, ZORBAX SB-C18, 250*4.6 mm, 5 μm and ACE Excel C18-AR, 150*4.6 mm, 3 μm respectively, and keep other conditions unchanged.

[0036] 2. Solution Preparation: The reference solution and the test solution are the same as those in Example 1, The spiked test solution at 100% limit level: the same as in Example 2.

[0037] 3. Detection 3.1 Inject the above three solutions into the chromatograph with the chromatographic column Waters XBridge C18, 150*4.5 mm, 3.5 μm respectively, record the chromatograms, and the results are shown in Figures 9 - 11 . Among them, Figure 9 , 10 , and 11 are the chromatograms of the reference solution, the test solution, and the spiked solution at 100% limit level after the test respectively. The results show that this chromatographic column cannot achieve good separation from the interference, the spiked recovery rate is abnormally high, and this method is not feasible. 3.2 Inject the reference solution and the test solution into the chromatograph with the chromatographic column ZORBAX SB-C18, 250*4.6 mm, 5 μm respectively, record the chromatograms, and the results are shown in Figures 12 - 13 . Figure 12 , 13 are the chromatograms of the reference solution and the test solution after the chromatographic column test respectively. The results show that this chromatographic column has no obvious improvement in separation, the main peak of the sample and the target compound cannot be completely separated, and the baseline is abnormal at the elution position of the target compound, and the method is not feasible.

[0038] 3.2 The reference solution and the test solution were respectively injected into a chromatograph with a ZORBAX SB-C18 column, 250*4.6 mm, 5 μm, and the chromatograms were recorded. The results are shown in Figures 12 - 13 . Figure 12 , 13 are the chromatograms of the reference solution and the test solution after the column test, respectively. The results show that: the column did not significantly improve the separation, the main peak of the sample and the target compound were not completely separated, and the baseline was abnormal at the elution position of the target compound. The method is not feasible.

[0039] 3.3 The above three solutions were respectively injected into a chromatograph with an ACE Excel C18-AR column, 150*4.6 mm, and the chromatograms were recorded. The results are shown in Figures 14 - 16 . Among them. Figure 14 , 15 and 16 are the chromatograms of the reference solution, the test solution, and the spiked solution at the 100% limit level, respectively. The results show that: for this column, the blank test sample (i.e., the unspiked test solution) and the spiked test solution had abnormal elution peaks and were not completely separated from the interference. The method is not feasible.

[0040] Summary of column screening: Ordinary C18 columns cannot achieve good separation of the target compound and the interference, and increasing the column length does not significantly improve the situation; in the present invention, a phenyl column and a column with a particle size of 3 μm (Luna phenyl-hexyl, 150*4.6 mm, 3 μm) are selected, achieving good separation from the interfering substances.

[0041] Any simple modification or variation of the technical solution of the present invention within the spirit of the present invention also belongs to the scope of the present invention.

Claims

1. A method for detecting nitrogen 10-nitrosofolinic acid in calcium folinate injection by liquid chromatography-mass spectrometry, characterized in that: The method comprises: using a phenyl column as a chromatographic column, mobile phase A is a 0.1% formic acid aqueous solution, mobile phase B is methanol, a gradient elution method is adopted, and an ion source is ESI (+).

2. The method of claim 1, wherein the phenyl column is a Phenomenex Luna 3μm Phenyl-hexyl100A, 150×4.6mm, 3μm or an equivalent chromatographic column.

3. The method according to claim 1, wherein the initial volume ratio of mobile phase A to mobile phase B is 70:

30.

4. The method according to claim 1, wherein the gradient elution is performed as follows: 。 5. The method of claim 1, wherein the flow rate is 0.5 ml / min. The method according to claim 1 , wherein the column temperature is 30° C.

7. The method according to claim 1, wherein the injection volume is 10 μl.

8. The method of claim 1, wherein the mass spectrometry parameters are as follows: 。 9. The method of claim 1, wherein the nitrogen 10-nitrosofolinic acid content in the test solution is calculated by the reference substance external standard method.

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