Analysis method for determining NTTP in sitagliptin phosphate

Abstract

The invention relates to the technical field of medicine analysis, and discloses an analysis method for measuring NTTP in sitagliptin phosphate, which comprises the following steps: S110: preparation of a test solution: taking a proper amount of sample, adding a first solvent for dissolving and diluting, filtering with a 0.25 [mu] m filter membrane, and taking the subsequent filtrate as the test solution; s120, preparing a reference substance solution: weighing a proper amount of NTTP reference substance, and adding a second solvent for dissolving to prepare a reference substance stock solution; precisely measuring a proper amount of reference substance stock solution, and diluting with a second solvent to prepare a reference substance solution; and S130, respectively injecting the test solution and the reference solution into an ultra-high performance liquid chromatograph-mass spectrometer, recording a chromatogram, and calculating the content of NTTP by peak area according to an external standard method. The method for analyzing the NTTP in the sitagliptin phosphate, established by the invention, is strong in specificity, high in stability, high in accuracy, high in precision and strong in durability, and can be used as a method for detecting the content of the NTTP in the sitagliptin phosphate.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical analysis, in particular to an analytical method for determining NTTP in sitagliptin phosphate. Background Art

[0002] Sitagliptin phosphate is a prescription medicine used to control high blood sugar in people with type 2 diabetes.

[0003] NTTP (Nitroso-stg-19), also known as 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine, is a nitrosamine-type genotoxic impurity that can directly or indirectly damage cell DNA, produce genotoxicity, and has the potential or tendency to cause cancer. NTTP is generated by the reaction of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-α]pyrazine, the starting material of sitagliptin phosphate, with nitrite in pharmaceutical excipients during the synthesis process. In order to prevent the adverse effects of treatment interruption on patients, the FDA did not recall sitagliptin, and stipulated that the temporary acceptable intake of NTTP can be up to 246.7 ng, and the lifetime daily intake is 37 ng. At present, there is an urgent need to control the content of NTTP in sitagliptin and develop appropriate detection methods and control measures. Currently, nitrosamine genotoxic impurities are mainly determined using liquid chromatography-tandem mass spectrometry, which has the advantages of high selectivity, good sensitivity, and low detection limit for trace compounds.

[0004] In the prior art, when analyzing NTTP, since a large amount of auxiliary materials exist in sitagliptin phosphate, the auxiliary materials cause great interference to the analysis results. Therefore, the present invention provides an analytical method for determining NTTP in sitagliptin phosphate. Summary of the invention

[0005] The purpose of the present invention is to provide an analytical method for detecting NTTP using an ultra-high performance liquid phase mass spectrometer, so as to accurately detect the content of NTTP in a product and realize quality control.

[0006] The present application embodiment provides a method for determining NTTP in sitagliptin phosphate, comprising the following steps:

[0007] S110: Preparation of test solution: Take an appropriate amount of sample, add the first solvent to dissolve and dilute, filter through a 0.25 μm filter membrane, and take the filtrate as the test solution;

[0008] S120: Preparation of reference substance solution: weigh an appropriate amount of NTTP reference substance, add a second solvent to dissolve and prepare a reference substance stock solution; accurately measure an appropriate amount of the reference substance stock solution, dilute it with the second solvent to prepare a reference substance solution;

[0009] S130: Inject the test solution and the reference solution into the ultra-high performance liquid phase mass spectrometer respectively, record the chromatogram, and calculate the NTTP content by the peak area according to the external standard method.

[0010] Furthermore, in S110, the first solvent is a mixture of 0.1% formic acid solution and 0.1% formic acid acetonitrile solution.

[0011] Furthermore, in S110, the volume ratio of the formic acid solution to the formic acid-acetonitrile solution is 95:5.

[0012] Furthermore, in S120, the second solvent is a 0.1% acetonitrile solution.

[0013] Further, in the S130, the liquid chromatography conditions are: using pentafluorophenyl as a filler, using 0.1% formic acid solution as mobile phase A; using 0.1% formic acid acetonitrile solution as mobile phase B, performing a gradient elution program, a column temperature of 35-45° C., a flow rate of 0.3-0.5 ml / min, and an injection volume of 5-20 μl;

[0014] Mass spectrometry conditions: detection was performed using a triple quadrupole tandem mass spectrometer, the ion source was an electrospray ionization (ESI) source, the positive ion scanning mode was the monitoring mode, the mass-to-charge ratio (m / z) 222→192 was selected as the detection ion pair.

[0015] Further, in S130, the gradient elution program is shown in the following table:

[0016]

[0017] Furthermore, in S130, the mass spectrometry conditions are shown in the following table:

[0018]

[0019] Furthermore, in S110, the test solution is a solution containing 25 ng of sitagliptin phosphate per 1 ml.

[0020] Furthermore, in S120, the reference substance stock solution is a solution containing 35 μg of NTTP per 1 ml; and the reference substance solution is a solution containing 7 ng of NTTP per 1 ml.

[0021] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0022] The NTTP analysis method in sitagliptin phosphate established by the present invention has strong specificity, high stability, high accuracy, high precision and strong durability, and can be used as a method for detecting the NTTP content in sitagliptin phosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0024] Figure 1 It is the characteristic spectrum of NTTP detection;

[0025] Figure 2 It is the NTTP linear diagram of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0030] Embodiment 1:

[0031] Chromatographic column: ACQUITY UPLC HSS PFP (2.1×100 mm, 1.8 μm)

[0032] Chromatographic conditions: column temperature 40°C; flow rate 0.4 ml / min; injection volume 10 μl; mobile phase A: 0.1% formic acid solution; mobile phase B: 0.1% formic acid acetonitrile solution; elution with gradient mobile phase, with the mobile phase volume ratio as 100%, the mobile phase gradient setting is as follows:

[0033] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 6 95 5 8 10 90 13 10 90 14 95 5 25 95 5

[0034] Mass spectrometry conditions: The mass spectrometer was used in electrospray positive ionization mode (ESI + ), multiple reaction monitoring (SRM) was performed, and the mass-to-charge ratio (m / z) 222→192 was selected as the detection ion pair. The specific mass spectrometry conditions were set as follows:

[0035]

[0036]

[0037] Specificity and system suitability:

[0038] Solution preparation:

[0039] Solvent: 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution (95:5).

[0040] Reference solution: Take about 3.5 mg of NTTP reference, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile, shake to dissolve NTTP, dilute to scale with acetonitrile, and shake well. Accurately measure 1 ml, place in a 25 ml volumetric flask, make up to volume with solvent, and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, make up to volume with solvent, and shake well.

[0041] Test solution: Accurately weigh about 250 mg of the product, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, filter, and obtain.

[0042] result:

[0043] Exclusive results:

[0044] Solution name Peak area Solvents Not detected

[0045] System suitability results:

[0046]

[0047]

[0048] The specificity and system suitability results met the requirements.

[0049] Limit of Detection and Limit of Quantitation:

[0050] Solution preparation:

[0051] Solvent: 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution (95:5).

[0052] Reference solution: Take about 3.5 mg of NTTP reference, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile, shake to dissolve NTTP, dilute to scale with acetonitrile, and shake well. Accurately measure 1 ml, place in a 25 ml volumetric flask, make up to volume with solvent, and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, make up to volume with solvent, and shake well.

[0053] Quantitative limit solution: Accurately measure 1 ml of the reference solution and place it in a 50 ml volumetric flask, add solvent to make up to volume, shake well, and the solution is ready.

[0054] Detection limit solution: Accurately measure 3 ml of the quantitative limit solution and place it in a 20 ml volumetric flask, add solvent to make up to volume, shake well, and the solution is ready.

[0055] result:

[0056] Detection limit results:

[0057] Concentration (ng / ml) Equivalent to test sample concentration (%) Equivalent to limit concentration (%) Signal-to-Noise Ratio (S / N) 0.04102 0.000000164 0.59 6.5

[0058] Limit of Quantitation Results:

[0059]

[0060] The detection limit and quantification limit experimental results can meet the detection requirements.

[0061] Linearity and Range:

[0062] Solution preparation:

[0063] Solvent: 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution (95:5).

[0064] Linear stock solution: Take about 3.5 mg of NTTP reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add acetonitrile, shake to dissolve NTTP, dilute acetonitrile to the mark, and shake well. Accurately measure 1 ml, place it in a 25 ml volumetric flask, add solvent to the fixed volume, shake well, and you have it.

[0065] Linear solution 1: Accurately measure 1 ml of linear stock solution, place it in a 10 ml volumetric flask, add solvent to dilute to the mark, and shake well. Accurately measure 1 ml of linear stock solution, place it in a 50 ml volumetric flask, add solvent to the volume, and shake well.

[0066] Linear solution 2: Accurately measure 1 ml of linear stock solution, place it in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0067] Linear solution 3: Accurately measure 1 ml of linear stock solution, place it in a 20 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0068] Linear solution 4: Accurately measure 1 ml of linear stock solution, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0069] Linear solution 5: Accurately measure 3 ml of linear stock solution, place in a 20 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0070] Linear solution 6: Accurately measure 2 ml of linear stock solution, place in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0071] result:

[0072] NTTP linear relationship:

[0073]

[0074]

[0075] NTTP has a good linear relationship.

[0076] Solution stability:

[0077] Solution preparation:

[0078] Solvent: 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution (95:5).

[0079] Reference solution: Take about 3.5 mg of NTTP reference, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile, shake to dissolve NTTP, dilute to scale with acetonitrile, and shake well. Accurately measure 1 ml, place in a 25 ml volumetric flask, make up to volume with solvent, and shake well. As reference stock solution. Accurately measure 1 ml of reference stock solution, place in a 20 ml volumetric flask, make up to volume with solvent, and shake well.

[0080] 100% spiked test solution: Accurately weigh about 250 mg of the product, place it in a 10 ml volumetric flask, add 1 ml of the reference stock solution, add solvent to dissolve and dilute to the scale, shake well, filter, and obtain.

[0081] result:

[0082] Reference solution stability results:

[0083] Time (h) NTTP peak area in reference solution 0 6615.640 7 6643.992 12.5 7112.009 16.7 7175.099 30.7 7437.263 0 6615.640 7 6643.992

[0084] 100% spiked test solution stability results:

[0085]

[0086]

[0087] Conclusion: Under room temperature, the reference solution is stable for 30.7 hours; the 100% spiked test solution is stable for 9.6 hours

[0088] Accuracy and Repeatability:

[0089] Solution preparation:

[0090] Solvent: 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution (95:5).

[0091] Reference solution: Take about 3.5 mg of NTTP reference, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile, shake to dissolve NTTP, dilute to scale with acetonitrile, and shake well. Accurately measure 1 ml, place in a 25 ml volumetric flask, make up to volume with solvent, and shake well. As the reference stock solution. Accurately measure 1 ml of the reference stock solution, place in a 20 ml volumetric flask, make up to volume with solvent, and shake well.

[0092] 50% spiked test solution: Accurately weigh about 250 mg of the product, place in a 10 ml volumetric flask, add 0.5 ml of reference stock solution, add solvent to dissolve and dilute to scale, shake well, filter, and prepare 3 portions in parallel.

[0093] 100% spiked test solution: Accurately weigh about 250 mg of the product, place in a 10 ml volumetric flask, add 1 ml of reference stock solution, dissolve and dilute to scale with solvent, shake well, filter, and prepare 6 portions in parallel. (The first 3 portions are used as 100% spiked test solution for accuracy, and all 6 portions are used as repeatability solutions).

[0094] 150% spiked test solution: Accurately weigh about 250 mg of the product, place in a 10 ml volumetric flask, add 1 ml of reference stock solution, add solvent to dissolve and dilute to scale, shake well, filter, and prepare 3 portions in parallel.

[0095] Accuracy results:

[0096]

[0097]

[0098] Reproducible results:

[0099]

[0100] The accuracy and repeatability results meet the test requirements.

[0101] Intermediate precision:

[0102] Solution preparation:

[0103] Solvent: 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution (95:5).

[0104] Reference solution: Take about 3.5 mg of NTTP reference, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile, shake to dissolve NTTP, dilute to scale with acetonitrile, and shake well. Accurately measure 1 ml, place in a 25 ml volumetric flask, make up to volume with solvent, and shake well. As reference stock solution. Accurately measure 1 ml of reference stock solution, place in a 20 ml volumetric flask, make up to volume with solvent, and shake well.

[0105] Intermediate precision solution: accurately weigh about 250 mg of this product, place it in a 10 ml volumetric bottle, add 1 ml of reference stock solution, add solvent to dissolve and dilute to the scale, shake well, filter, and prepare 6 copies in parallel.

[0106] result:

[0107] Intermediate precision results:

[0108]

[0109] The intermediate precision results meet the test requirements and have good precision.

[0110] Durability:

[0111] Solution preparation:

[0112] Solvent: 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution (95:5).

[0113] Reference solution: Take about 3.5 mg of NTTP reference, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile, shake to dissolve NTTP, dilute to scale with acetonitrile, and shake well. Accurately measure 1 ml, place in a 25 ml volumetric flask, make up to volume with solvent, and shake well. As reference stock solution. Accurately measure 1 ml of reference stock solution, place in a 20 ml volumetric flask, make up to volume with solvent, and shake well.

[0114] Sensitivity solution: Accurately measure 1 ml of the reference solution and place it in a 20 ml volumetric flask, add solvent to make up the volume, shake well, and the solution is ready.

[0115] 100% spiked test solution: Accurately weigh about 250 mg of the product, place it in a 10 ml volumetric flask, add 1 ml of the reference stock solution, add solvent to dissolve and dilute to the scale, shake well, filter, and obtain.

[0116] result:

[0117] Solvent durability results:

[0118]

[0119]

[0120] Sensitivity Solution Durability Results:

[0121]

[0122] Reference solution durability results:

[0123]

[0124] Reference solution durability results:

[0125]

[0126]

[0127] The durability results meet the testing requirements.

[0128] NTTP detection characteristic spectrum see Figure 1 .

[0129] In summary, the NTTP analysis method in sitagliptin phosphate established in the present invention has strong specificity, high stability, high accuracy, high precision and strong durability, and can be used as a method for detecting the NTTP content in sitagliptin phosphate.

[0130] The above are only preferred embodiments of the present invention. It should be pointed out that researchers in this field can make several modifications and changes without departing from the creative concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for determining NTTP in sitagliptin phosphate, characterized in that: The following steps are involved: S110: Preparation of test solution: Take an appropriate amount of sample, add the first solvent to dissolve and dilute, filter through a 0.25 μm filter membrane, and take the filtrate as the test solution; S120: Preparation of reference substance solution: weigh an appropriate amount of NTTP reference substance, add a second solvent to dissolve and prepare a reference substance stock solution; accurately measure an appropriate amount of the reference substance stock solution, dilute it with the second solvent to prepare a reference substance solution; S130: Inject the test solution and the reference solution into the ultra-high performance liquid phase mass spectrometer respectively, record the chromatogram, and calculate the NTTP content by the peak area according to the external standard method.

2. A method for determining NTTP in sitagliptin phosphate according to claim 1, characterized in that: In the S110, the first solvent is a mixture of 0.1% formic acid solution and 0.1% formic acid acetonitrile solution.

3. A method for determining NTTP in sitagliptin phosphate according to claim 2, characterized in that: In the S110, the volume ratio of the formic acid solution to the formic acid acetonitrile solution is 95:

5.

4. A method for determining NTTP in sitagliptin phosphate according to claim 3, characterized in that: In the S120, the second solvent is a 0.1% acetonitrile solution.

5. A method for determining NTTP in sitagliptin phosphate according to claim 4, characterized in that: In the S130, the liquid chromatography conditions are: using pentafluorophenyl as a filler, using 0.1% formic acid solution as mobile phase A; using 0.1% formic acid acetonitrile solution as mobile phase B, performing a gradient elution program, a column temperature of 35-45° C., a flow rate of 0.3-0.5 ml / min, and an injection volume of 5-20 μl; Mass spectrometry conditions: detection was performed using a triple quadrupole tandem mass spectrometer, the ion source was an electrospray ionization (ESI) source, the positive ion scanning mode was the monitoring mode, the mass-to-charge ratio (m / z) 222→192 was selected as the detection ion pair.

6. A method for determining NTTP in sitagliptin phosphate according to claim 5, characterized in that: In the S130, the gradient elution program is shown in the following table:

7. A method for determining NTTP in sitagliptin phosphate according to claim 5, characterized in that: In S130, the mass spectrometry conditions are shown in the following table: 。 8. A method for determining NTTP in sitagliptin phosphate according to claim 1, characterized in that: In the S110, the test solution is a solution containing 25 ng of sitagliptin phosphate per 1 ml.

9. A method for determining NTTP in sitagliptin phosphate according to claim 1, characterized in that: In the S120, the reference substance stock solution is a solution containing 35 μg of NTTP per 1 ml; the reference substance solution is a solution containing 7 ng of NTTP per 1 ml.

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