Detection method of hydrazine compound and application thereof

Abstract

The invention provides a hydrazine compound detection method and application thereof, and belongs to the technical field of pharmaceutical analysis. The detection method comprises the following steps: dissolving a to-be-detected sample with water, mixing the to-be-detected sample with a derivatization reagent, carrying out a derivatization reaction on the hydrazine compounds in the to-be-detected sample and the derivatization reagent to generate a derivative product, detecting the content of the derivative product by adopting a high performance liquid chromatography-mass spectrometry method, and calculating the content of the hydrazine compounds. The method can simultaneously detect chloroacethydrazide and trifluoroacethydrazide, and has the advantages of low detection limit concentration and quantitation limit concentration, high sensitivity, good repeatability, high accuracy and good separation effect.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a detection method for hydrazine compounds and application thereof. Background Art

[0002] Chloroacetylhydrazine and trifluoroacetylhydrazine are impurities that may exist in the hypoglycemic drug sitagliptin phosphate. Hydrazide has a genotoxic warning structure. With the increasing attention paid to drug safety, the detection of extremely trace impurities remaining in drugs has gradually been included in drug quality standards or corporate internal control standards. Therefore, it is necessary to establish a detection method for chloroacetylhydrazine and apply it to the detection method of trace chloroacetylhydrazine that may exist in sitagliptin phosphate or its starting materials or intermediates to ensure the safety of clinical patients' medication.

[0003] At present, there is a patent reporting the detection of chloroacetylhydrazine in sitagliptin phosphate by high performance liquid chromatography elution in HILIC mode. This method has poor sensitivity and is not sufficient to detect chloroacetylhydrazine at the ppm level in the sample. Moreover, chloroacetylhydrazine and trifluoroacetylhydrazine are small molecular compounds with high polarity and poor retention on the C18 column, making it difficult to achieve a good separation effect.

[0004] In addition, there is currently no report on a method for simultaneously detecting chloroacetylhydrazine and trifluoroacetylhydrazine.

[0005] Therefore, there is an urgent need for a method with high sensitivity, good separation effect, and the ability to simultaneously detect chloroacetylhydrazine and trifluoroacetylhydrazine. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a detection method and application of hydrazine compounds.

[0007] In a first aspect, the present invention provides a method for detecting hydrazine compounds.

[0008] A method for detecting hydrazine compounds comprises: dissolving a sample to be tested in water and then mixing it with a derivatization reagent, so that the hydrazine compounds in the sample to be tested react with the derivatization reagent to produce a derivative product, then using high performance liquid chromatography-mass spectrometry to detect the content of the derivative product, and then calculating the content of the hydrazine compounds.

[0009] In some embodiments, the present application adopts an external standard method to calculate the content of hydrazine compounds.

[0010] In some embodiments, the hydrazine compound is a hydrazide compound.

[0011] In some embodiments, the hydrazide compound includes at least one of chloroacetohydrazide or trifluoroacetohydrazide.

[0012] In some embodiments, the derivatizing agent comprises benzaldehyde.

[0013] In some embodiments, the derivatizing agent comprises benzaldehyde and an acid.

[0014] In some embodiments, the derivatization reagent is a solution containing benzaldehyde, an acid, and a solvent.

[0015] In some embodiments, the acid comprises at least one of formic acid, acetic acid, or phosphoric acid.

[0016] In some embodiments, the solvent comprises at least one of acetonitrile, methanol, or water.

[0017] In some embodiments, the concentration of benzaldehyde in the derivatization reagent is 1.0% vol-4.0% vol. In some embodiments, the concentration of benzaldehyde in the derivatization reagent is 1.0% vol, 2.0% vol, 3.0% vol or 4.0% vol. In some embodiments, the concentration of benzaldehyde in the derivatization reagent is 2.0% vol.

[0018] In some embodiments, the concentration of the acid in the derivatization reagent is 0.5% vol-2.0% vol. In some embodiments, the concentration of the acid in the derivatization reagent is 0.5% vol, 1.0% vol, 1.5% vol or 2.0% vol. In some embodiments, the concentration of the acid in the derivatization reagent is 1.0% vol.

[0019] In some embodiments, 10 mg to 30 mg of the sample to be tested is added per 1 ml of the derivatization reagent. In some embodiments, 10 mg, 15 mg, 20 mg, 25 mg or 30 mg of the sample to be tested is added per 1 ml of the derivatization reagent. In some embodiments, 20 mg of the sample to be tested is added per 1 ml of the derivatization reagent.

[0020] In some embodiments, 5 mg to 50 mg of the sample to be tested is dissolved per 1 ml of water. In some embodiments, 10 mg to 30 mg of the sample to be tested is dissolved per 1 ml of water. In some embodiments, 15 mg to 25 mg of the sample to be tested is dissolved per 1 ml of water. In some embodiments, 20 mg of the sample to be tested is dissolved per 1 ml of water. In some embodiments, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg of the sample to be tested is dissolved per 1 ml of water.

[0021] In some embodiments, the reaction temperature of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent is 50-65° C. In some embodiments, the reaction temperature of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent is 50° C., 55° C., 60° C. or 65° C. In some embodiments, the reaction temperature of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent is 60° C.

[0022] In some embodiments, the reaction time of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent is 1.5h-2.5h. In some embodiments, the reaction time of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent is 1.5h, 2.0h or 2.5h. In some embodiments, the reaction time of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent is 2.0h.

[0023] In some embodiments, the detection method further includes the preparation and detection of a reference solution, and the preparation method of the reference solution includes: mixing a hydrazine compound solution with a derivatization reagent to generate a derivatization reaction to obtain the reference solution.

[0024] In some embodiments, the concentration of the hydrazine compound in the hydrazine compound solution is 0.1 μg / ml-0.5 μg / ml. In some embodiments, the concentration of the hydrazine compound in the hydrazine compound solution is 0.2 μg / ml-0.4 μg / ml. In some embodiments, the concentration of the hydrazine compound in the hydrazine compound solution is 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml or 0.5 μg / ml.

[0025] In some embodiments, the reaction temperature of the derivatization reaction in the method for preparing the reference solution is 50° C.-65° C. In some embodiments, the reaction temperature of the derivatization reaction in the method for preparing the reference solution is 50° C., 55° C., 60° C. or 65° C. In some embodiments, the reaction temperature of the derivatization reaction in the method for preparing the reference solution is 60° C.

[0026] In some embodiments, the reaction time of the derivatization reaction in the method for preparing the reference solution is 1.5 h to 2.5 h. In some embodiments, the reaction time of the derivatization reaction in the method for preparing the reference solution is 2 h. In some embodiments, the reaction time of the derivatization reaction in the method for preparing the reference solution is 1.5 h, 2.0 h or 2.5 h.

[0027] In some embodiments, the volume ratio of the hydrazine compound solution to the derivatization reagent in the preparation method of the reference solution is 1:1-1:2. In some embodiments, the volume ratio of the hydrazine compound solution to the derivatization reagent in the preparation method of the reference solution is 1:1.

[0028] In some embodiments, the reaction temperature and reaction time of the derivatization reaction between the hydrazine compound in the test sample and the derivatization reagent are respectively the same as the reaction temperature and reaction time of the derivatization reaction in the preparation method of the reference solution.

[0029] In some embodiments, the hydrazine compound includes chloroacetylhydrazine and trifluoroacetylhydrazine, and the preparation method of the reference solution includes: mixing a hydrazine compound solution containing 0.1μg / ml-0.5μg / ml chloroacetylhydrazine and 0.1μg / ml-0.5μg / ml trifluoroacetylhydrazine with a derivatization reagent, reacting at 50-65°C for 2.0h to obtain the reference solution, wherein the concentration is a volume ratio of the hydrazine compound solution to the derivatization reagent of 1:1-1:2.

[0030] In some embodiments, the hydrazine compound includes chloroacetylhydrazine and trifluoroacetylhydrazine, and the preparation method of the reference solution includes: mixing a hydrazine compound solution containing 0.2 μg / ml chloroacetylhydrazine and 0.2 μg / ml trifluoroacetylhydrazine with a derivatization reagent, reacting at 60° C. for 2 hours to obtain the reference solution, wherein the concentration is a volume ratio of the hydrazine compound solution to the derivatization reagent of 1:1.

[0031] In some embodiments, the solvent of the hydrazine compound solution is an organic solvent and / or water.

[0032] In some embodiments, the preparation method of the hydrazine compound solution includes: dissolving chloroacetylhydrazine with 0.05%vol-0.20%vol or 0.10%vol acid aqueous solution and then diluting with an organic solvent to obtain a chloroacetylhydrazine stock solution; diluting trifluoroacetylhydrazine with an organic solvent to obtain a trifluoroacetylhydrazine stock solution; mixing the chloroacetylhydrazine stock solution and the trifluoroacetylhydrazine stock solution, and diluting with an organic solvent to obtain a reference substance stock solution, and then diluting the reference substance stock solution with water to obtain the hydrazine compound solution.

[0033] In some embodiments, the acid in the aqueous acid solution is formic acid.

[0034] In some embodiments, the concentration of chloroacetylhydrazine in the chloroacetylhydrazine stock solution is 0.5 mg / ml-2.5 mg / ml. In some embodiments, the concentration of chloroacetylhydrazine in the chloroacetylhydrazine stock solution is 1.0 mg / ml.

[0035] In some embodiments, the concentration of trifluoroacetylhydrazine in the trifluoroacetylhydrazine stock solution is 0.5 mg / ml-2.5 mg / ml. In some embodiments, the concentration of trifluoroacetylhydrazine in the trifluoroacetylhydrazine stock solution is 1.0 mg / ml.

[0036] In some embodiments, the concentration of chloroacetylhydrazine in the reference substance stock solution is 0.01 mg / ml-0.05 mg / ml. In some embodiments, the concentration of chloroacetylhydrazine in the reference substance stock solution is 0.02 mg / ml.

[0037] In some embodiments, the concentration of trifluoroacetylhydrazine in the reference stock solution is 0.01 mg / ml-0.05 mg / ml. In some embodiments, the concentration of trifluoroacetylhydrazine in the reference stock solution is 0.02 mg / ml.

[0038] In some embodiments, the organic solvent is acetonitrile.

[0039] In some embodiments, the high performance liquid chromatography method uses a C18 chromatographic column for detection, preferably a Waters HSS T3 column was used for detection.

[0040] In some embodiments, the Waters The column length of HSS T3 chromatographic column is 100mm-250mm.

[0041] In some embodiments, the Waters The inner diameter of the HSS T3 chromatographic column is 2 mm to 6 mm. The inner diameter of the HSS T3 chromatographic column is 3 mm to 5 mm. The inner diameter of HSS T3 columns is 3.9mm-4.6mm.

[0042] In some embodiments, the Waters The particle size of the filler of the HSS T3 chromatographic column is 1.8 μm-5 μm. In some embodiments, the Waters The particle size of the filler of the HSS T3 chromatographic column is 2 μm-3 μm. The particle size of the packing material of the HSS T3 column is 2.5 μm.

[0043] In some embodiments, the high performance liquid chromatography method uses a mixture of acetonitrile and 0.05% vol-0.20% vol formic acid aqueous solution in a volume ratio of 2.5:97.5 to 10.0:90.0 as mobile phase A, acetonitrile as mobile phase B, and performs isocratic elution or gradient elution. In some embodiments, the high performance liquid chromatography method uses a mixture of acetonitrile and 0.05% vol, 0.10% vol, 0.15% vol or 0.20% vol formic acid aqueous solution in a volume ratio of 2.5:97.5, 4.0:96.0, 5.0:95.0, 6.0:94.0, 7.0:93.0, 8.0:92.0, 9.0:91.0 or 10.0:90.0 as mobile phase A, acetonitrile as mobile phase B, and performs isocratic elution or gradient elution. In some embodiments, the HPLC method uses a mixture of acetonitrile and 0.10% vol formic acid aqueous solution in a volume ratio of 5.0:95.0 as mobile phase A, acetonitrile as mobile phase B, and performs isocratic elution or gradient elution.

[0044] In some embodiments, the isocratic elution is performed using a volume ratio of mobile phase A to mobile phase B of 75:25 to 85:15. In some embodiments, the isocratic elution is performed using a volume ratio of mobile phase A to mobile phase B of 75:25, 78:22, 80:20, 82:18 or 85:15. In some embodiments, the isocratic elution is performed using a volume ratio of mobile phase A to mobile phase B of 78:22 to 82:18. In some embodiments, the isocratic elution is performed using a volume ratio of mobile phase A to mobile phase B of 80:20.

[0045] In some embodiments, the isocratic elution time is 12 min to 25 min. In some embodiments, the isocratic elution time is 25 min.

[0046] In some embodiments, the elution program of the gradient elution is:

[0047] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75～85 25～15 9～11 55～65 45～35 12～25 55～65 45～35

[0048] In some embodiments, the elution program of the gradient elution is:

[0049] Time (min) Mobile phase A (%) Mobile phase B (%) 0 78～82 22～18 9～11 58～62 42～38 12～25 58～62 42～38 .

[0050] In some embodiments, the elution program of the gradient elution is:

[0051] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 9～11 60 40 12～25 60 40 .

[0052] In some embodiments, the elution program of the gradient elution is:

[0053] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 9 60 40 12～25 60 40 .

[0054] In some embodiments, the flow rate of the HPLC is 0.8 ml / min-1.2 ml / min. In some embodiments, the flow rate of the HPLC is 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min or 1.2 ml / min. In some embodiments, the flow rate of the HPLC is 0.9 ml / min-1.1 ml / min. In some embodiments, the flow rate of the HPLC is 1.0 ml / min.

[0055] In some embodiments, the column temperature of the HPLC is 15°C-40°C. In some embodiments, the column temperature of the HPLC is 15°C, 20°C, 25°C, 30°C, 35°C or 40°C. In some embodiments, the column temperature of the HPLC is 25°C-35°C. In some embodiments, the column temperature of the HPLC is 28°C-32°C. In some embodiments, the column temperature of the HPLC is 29°C-31°C. In some embodiments, the column temperature of the HPLC is 30°C.

[0056] In some embodiments, the injection volume of the HPLC is 5 μL-50 μL. In some embodiments, the injection volume of the HPLC is 10 μL-20 μL. In some embodiments, the injection volume of the HPLC is 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL or 50 μL.

[0057] In some embodiments, the elution procedure of the HPLC method further includes a post-run time of 2-10 min.

[0058] In some embodiments, the ion source of the mass spectrometry is an electrospray ion source.

[0059] In some embodiments, the mass spectrometry scan mode is SIM m / z=197(+); m / z=215(-).

[0060] In some embodiments, the mass spectrometry acquisition time is 4 min-25 min. In some embodiments, the mass spectrometry acquisition time is 4 min-20 min. In some embodiments, the mass spectrometry acquisition time is 4 min-15 min. In some embodiments, the mass spectrometry acquisition time is 4 min-12 min.

[0061] In some embodiments, the drying gas temperature of the mass spectrometry is 300° C.-350° C. In some embodiments, the drying gas temperature of the mass spectrometry is 300° C., 310° C., 320° C., 330° C., 340° C., or 350° C. In some embodiments, the drying gas temperature of the mass spectrometry is 300° C.

[0062] In some embodiments, the mass spectrometry drying gas flow rate is 8 L / min-12 L / min. In some embodiments, the mass spectrometry drying gas flow rate is 8 L / min, 9 L / min, 10 L / min, 11 L / min or 12 L / min. In some embodiments, the mass spectrometry drying gas flow rate is 12 L / min.

[0063] In some embodiments, the nebulizer gas pressure of the mass spectrometry is 30psi-50psi. In some embodiments, the nebulizer gas pressure of the mass spectrometry is 30psi, 35psi, 40psi, 45psi or 50psi. In some embodiments, the nebulizer gas pressure of the mass spectrometry is 45psi.

[0064] In some embodiments, the capillary voltage of the mass spectrometry is 3500 V (+), 3500 V (-). In some embodiments, the capillary voltage of the mass spectrometry is 4000 V (+), 4000 V (-). In some embodiments, the capillary voltage of the mass spectrometry is 4500 V (+), 4500 V (-).

[0065] In some embodiments, the fragmentation voltage of the mass spectrometry is 70 V to 90 V. In some embodiments, the fragmentation voltage of the mass spectrometry is 70 V, 75 V, 80 V, 85 V, or 90 V.

[0066] In some embodiments, the detection method includes: dissolving the sample to be tested in water and mixing it with a derivatization reagent, allowing the hydrazine compound in the sample to be tested to undergo a derivatization reaction with the derivatization reagent at 60°C for 2 hours to produce a derivative product, wherein the derivatization reagent is an acetonitrile solution containing 2.0% vol benzaldehyde and 1.0% vol formic acid; then using high performance liquid chromatography-mass spectrometry to detect the content of the derivative product and calculate the content of the hydrazine compound; the high performance liquid chromatography method uses Waters HSS T3 column was used for detection. The HSS T3 chromatographic column has a column length of 100 mm, an inner diameter of 4.6 mm, and a filler particle size of 2.5 μm. The high performance liquid chromatography method uses a mixture of acetonitrile and 0.10% vol formic acid aqueous solution in a volume ratio of 5.0:95.0 as mobile phase A, and acetonitrile as mobile phase B for gradient elution. The elution procedure of the gradient elution is:

[0067] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 9 60 40 12 60 40 ;

[0068] The flow rate of the HPLC method is 1.0 ml / min;

[0069] The column temperature of the HPLC method is 30°C;

[0070] The elution procedure of the high performance liquid chromatography also includes a post-run time of 5 min;

[0071] The mass spectrometry method adopts a liquid chromatography-mass spectrometer; the ion source of the mass spectrometry method is an electrospray ion source; the scanning mode of the mass spectrometry method is SIM m / z=197(+); m / z=215(-); the acquisition time of the mass spectrometry method is 4min-12min; the drying gas temperature of the mass spectrometry method is 300°C; the drying gas flow rate of the mass spectrometry method is 12L / min; the nebulizing gas pressure of the mass spectrometry method is 45psi; the capillary voltage of the mass spectrometry method is 3500V(+), 3500V(-), or the capillary voltage of the mass spectrometry method is 4500V(+), 4500V(-); the fragmentation voltage of the mass spectrometry method is 70V-90V.

[0072] In a second aspect, the present invention provides applications of the aforementioned detection method.

[0073] A method for detecting the content of chloroacetylhydrazine and / or trifluoroacetylhydrazine in sitagliptin or its salt, a preparation containing sitagliptin or its salt, (3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine or its salt, or an intermediate compound for synthesizing sitagliptin or its salt.

[0074] Beneficial Effects

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] The present invention solves the problem that the current detection limits of chloroacetylhydrazine and trifluoroacetylhydrazine are not reached by using a derivatized HPLC-MS method, and fills the gap in the method for simultaneously detecting chloroacetylhydrazine and trifluoroacetylhydrazine. The detection method of the present application has low detection limit concentration and quantitative limit concentration, high sensitivity, good repeatability, high accuracy and good separation effect.

[0077] Terminology

[0078] The term "room temperature" means ambient temperature, which refers to a temperature between about 10°C and about 30°C, or between about 20°C and 30°C, or about 25°C. In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0079] The term "v / v" means volume ratio.

[0080] The term "% vol" means volume percentage.

[0081] The term "wt%" means percent by mass.

[0082] In the following, all numbers disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. Whenever a number with a value of N is disclosed, any number with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15% or N+ / -20% will be explicitly disclosed, where "+ / -" means plus or minus.

[0083] The structural formula of chloroacetylhydrazine described in the present invention is The molecular formula is C2H5ClN2O.

[0084] The structural formula of trifluoroacetylhydrazine of the present invention is The molecular formula is C2H3F3N2O. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 This is the chromatogram of sensitivity solution No. 1 in Example 3.

[0086] Figure 2 This is the chromatogram of the reference solution in Example 7-1.

[0087] Figure 3 This is the chromatogram of the reference solution in Example 7-2.

[0088] Figure 4This is the chromatogram of the reference solution in Example 7-3. DETAILED DESCRIPTION

[0089] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further describe the present invention in detail.

[0090] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0091] Unless otherwise specified, the test conditions used in the examples or comparative examples of the present invention are as follows:

[0092] Chromatographic conditions:

[0093] Chromatographic column: Waters HSS T3 column, 4.6 mm × 100 mm, 2.5 μm;

[0094] Detector: Liquid chromatography mass spectrometry detector;

[0095] Ion source: ESI;

[0096] Scan mode: SIM m / z = 197 (+); m / z = 215 (-);

[0097] Flow rate: 1.0ml / min;

[0098] Column temperature: 30°C;

[0099] Injection volume: 10 μl;

[0100] Mobile phase A: 5% acetonitrile-95% (0.1% formic acid in water);

[0101] Mobile phase B: acetonitrile;

[0102] Elution ratio:

[0103] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 10 60 40 12～15 60 40

[0104] Mass spectrometry parameters:

[0105]

[0106]

[0107] Running time: 12 minutes;

[0108] Post-run: 5min;

[0109] MS acquisition time: 4-12 min.

[0110] Unless otherwise specified, the test sample described in the following examples or comparative examples is 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride (abbreviated as TTTP, which is the starting material of sitagliptin phosphate), and its structural formula is:

[0111] In the following examples or comparative examples, the retention time is retained to two decimal places. In the following weighing process, the error within 10% of the theoretical weight is acceptable. For example, when weighing about 50 mg of chloroacetylhydrazine, an error range of no more than 10% is acceptable when actually weighing.

[0112] In the examples of the present invention, "N / A" or "--" indicates no peak or no data or not applicable.

[0113] The calculation formula for the content of chloroacetylhydrazine or trifluoroacetylhydrazine in the test sample is:

[0114]

[0115] Where: A T is the peak area of ​​the derivative product measured in the test solution;

[0116] A S The reference solution was injected three times continuously to obtain the average peak area of ​​the derivative product;

[0117] W T is the amount of the test sample when preparing the test solution, mg;

[0118] W S The mass of the impurity reference substance (chloroacetylhydrazine or trifluoroacetylhydrazine) to be measured when preparing the reference solution, mg;

[0119] D T is the dilution factor of the test solution;

[0120] D S is the dilution multiple of the reference solution;

[0121] P is the content of the reference substance, %.

[0122] Example 1: System suitability investigation

[0123] Experimental procedures

[0124] Derivatization reagent: Take 1.0 ml of benzaldehyde and 0.5 ml of formic acid in a 50 ml volumetric flask, dissolve and dilute with acetonitrile, shake well, and obtain;

[0125] Blank solution: Mix 1.0 ml ultrapure water and 1.0 ml derivatization reagent, and react in a 60°C water bath for 2 h to obtain the blank solution.

[0126] Chloroacetylhydrazine stock solution: weigh about 50 mg of chloroacetylhydrazine accurately, put it into a 50 ml volumetric flask, dissolve it with 5.0 ml of 0.1% formic acid water, and then make up to volume with acetonitrile, shake well, and you have it;

[0127] Trifluoroacetylhydrazine stock solution: Weigh about 50 mg of trifluoroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it in acetonitrile and make up to volume, and shake well;

[0128] Reference substance stock solution: Pipette 1.0 ml of chloroacetylhydrazine stock solution and 1.0 ml of trifluoroacetylhydrazine stock solution into a 50 ml volumetric flask, dilute to volume with acetonitrile, and shake well;

[0129] Hydrazine compound solution: Pipette 1.0 ml of reference stock solution into a 100 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0130] Reference solution: Take 1.0 ml of hydrazine compound solution and 1.0 ml of derivatization reagent, mix, and react in a 60°C water bath for 2 hours to obtain the reference solution.

[0131] Detection limit stock solution: Pipette 3.0 ml of hydrazine compound solution into a 10 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0132] Detection limit solution: Take 1.0 ml of detection limit stock solution and 1.0 ml of derivatization reagent, mix, and react in a 60℃ water bath for 2 h to obtain the solution;

[0133] Take 1 injection of blank solution and detection limit solution, 3 injections of reference solution (reference solution-1, reference solution-2 and reference solution-3), and record the chromatogram. The experimental results of system suitability investigation are shown in Table A below.

[0134] Table A: System suitability investigation results

[0135]

[0136] in conclusion:

[0137] 1. The signal-to-noise ratios (S / N) of the chromatographic peaks of chloroacetylhydrazine derivatives and trifluoroacetylhydrazine derivatives in the detection limit solution were 23.7 and 106.5, respectively (acceptable standard: S / N ≥ 3);

[0138] 2. The separation degree between the chloroacetylhydrazine derivative product and the adjacent chromatographic peaks in the first injection of the reference solution was 1.8, and the separation degree between the trifluoroacetylhydrazine derivative product and the adjacent chromatographic peaks was 14.3 (acceptable standard: separation degree with adjacent chromatographic peaks ≥1.5). The RSDs of the peak areas of the chloroacetylhydrazine derivative product and the trifluoroacetylhydrazine derivative product of the reference solution after three consecutive injections were 3.63% and 1.18%, respectively (acceptable standard: RSD≤15.0%).

[0139] In summary, the system applicability of the method provided by the present invention meets the detection requirements.

[0140] Example 2: Investigation of specificity and accuracy

[0141] Experimental procedures

[0142] Derivatization reagent: Take 1.0 ml of benzaldehyde and 0.5 ml of formic acid in a 50 ml volumetric flask, dissolve and dilute with acetonitrile, shake well, and obtain;

[0143] Blank solution: Mix 1.0 ml ultrapure water and 1.0 ml derivatization reagent, and react in a 60°C water bath for 2 h to obtain the blank solution.

[0144] Chloroacetylhydrazine stock solution: weigh about 50 mg of chloroacetylhydrazine accurately, put it into a 50 ml volumetric flask, dissolve it with 5.0 ml of 0.1% formic acid water, and then make up to volume with acetonitrile, shake well, and you have it;

[0145] Trifluoroacetylhydrazine stock solution: Weigh about 50 mg of trifluoroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it in acetonitrile and make up to volume, and shake well;

[0146] Reference substance stock solution: Pipette 1.0 ml of chloroacetylhydrazine stock solution and 1.0 ml of trifluoroacetylhydrazine stock solution into a 50 ml volumetric flask, dilute to volume with acetonitrile, and shake well;

[0147] Hydrazine compound solution: Pipette 1.0 ml of reference stock solution into a 100 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0148] Reference solution: Take 1.0 ml of hydrazine compound solution and 1.0 ml of derivatization reagent, mix, and react in a 60°C water bath for 2 hours to obtain the reference solution.

[0149] Test sample spike solution: Take about 20 mg of the test sample, accurately weigh it into a 10 ml EP tube, dissolve it with 1.0 ml of hydrazine compound solution, add 1.0 ml of derivatization reagent, react in a 60 ° C water bath for 2 hours to obtain a 100% test sample spike solution, prepare 3 copies in parallel;

[0150] Preparation of test solution: weigh about 20 mg of the test sample, add 1.0 ml of ultrapure water to dissolve, and 1.0 ml of derivatization reagent, react in a 60°C water bath for 2 h.

[0151] Take each of the above solutions according to the chromatographic conditions described in the method, inject one needle into each portion, and record the chromatogram. Report the retention time, peak area and separation degree of each impurity derivative product to be tested in the blank solution, reference solution, test solution, and test solution spiked with the test product;

[0152] Calculate the spike recovery (R value), the average value of the R value and its RSD value. The calculation formula of the R value is as follows:

[0153]

[0154] Where: A T+S is the peak area of ​​each impurity-derived product in the test sample spiked solution;

[0155] A T is the peak area of ​​each impurity-derived product in the test solution;

[0156] A S It is the average of the peak areas of each impurity derivative product in 3 reference solutions;

[0157] W T is the weight of the test sample in the test solution, mg;

[0158] W T+S is the weight of the test sample in the test sample spike solution, mg.

[0159] The specificity results are shown in Table B and the accuracy results are shown in Table C.

[0160] Table B: Specificity results

[0161]

[0162] in conclusion:

[0163] 1. The blank solution has no interference with the impurity derivatives in the reference solution, the test solution, and the test spiked solution, and meets the acceptable standard requirements;

[0164] 2. The minimum separation between each impurity derivative product peak and the adjacent chromatographic peak in the reference solution, test solution, and test spiked solution is 6.8, which meets the acceptance standard (acceptable standard: the separation between each impurity derivative product peak and the adjacent chromatographic peak ≥1.5);

[0165] 3. Chloroacetylhydrazine was not detected in the test solution. The peak retention time of trifluoroacetylhydrazine derivative products in the test solution was consistent with that in the reference solution. The peak retention time of each impurity derivative product to be tested in the test spiked solution was consistent with that in the reference solution, which met the acceptable standard.

[0166] 4. Chloroacetylhydrazine was not detected in the test solution. Compared with the test solution, the peak areas of chloroacetylhydrazine derivatives and trifluoroacetylhydrazine derivatives in the test spiked solution increased significantly, which met the acceptable standards.

[0167] In summary, the detection method provided by the present invention has good specificity.

[0168] Table C: Accuracy Results

[0169]

[0170] in conclusion:

[0171] 1. The R values ​​of chloroacetylhydrazine derivatives and trifluoroacetylhydrazine derivatives in the test sample spiked solution ranged from 94.6% to 101.8% and 99.9% to 100.9% respectively (the acceptable standard was between 70.0% and 130.0%).

[0172] 2. The RSD (n=3) values ​​of the R values ​​of chloroacetylhydrazine derivatives and trifluoroacetylhydrazine derivatives in the test sample spiked solution were 3.82% and 0.54%, respectively (acceptable standard: ≤15.0%).

[0173] In summary, the detection method provided by the present invention has good accuracy.

[0174] Example 3: Sensitivity study

[0175] Experimental procedures

[0176] Derivatization reagent: Take 1.0ml benzaldehyde + 0.5ml formic acid in a 50ml volumetric flask, dissolve and dilute with acetonitrile, shake well, and you have it;

[0177] Blank solution: 1.0ml ultrapure water + 1.0ml derivatization reagent in a 10ml EP tube, react in a 60℃ water bath for 2h to obtain the blank solution;

[0178] Chloroacetylhydrazine stock solution: weigh about 50 mg of chloroacetylhydrazine accurately, put it into a 50 ml volumetric flask, dissolve it with 5.0 ml of 0.1% formic acid water, and then make up to volume with acetonitrile, shake well, and you have it;

[0179] Trifluoroacetylhydrazine stock solution: Weigh about 50 mg of trifluoroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it in acetonitrile and make up to volume, and shake well;

[0180] Reference substance stock solution: Pipette 1.0 ml of chloroacetylhydrazine stock solution + 1.0 ml of trifluoroacetylhydrazine stock solution into a 50 ml volumetric flask, dilute to volume with acetonitrile, and shake well;

[0181] Hydrazine compound solution: Pipette 1.0 ml of reference stock solution into a 100 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0182] Reference solution: 1.0 ml hydrazine compound solution + 1.0 ml derivatization reagent in a 10 ml EP tube, react in a 60°C water bath for 2 hours to obtain the solution;

[0183] Sensitivity stock solution: Pipette 3.0 ml of hydrazine compound solution into a 10 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0184] Sensitivity solution: 1.0ml sensitivity stock solution + 1.0ml derivatization reagent into a 10ml EP tube, react in a 60℃ water bath for 2h to obtain the solution;

[0185] Take a blank solution and inject 1 needle, and then inject 3 needles of the sensitivity solution, and record the chromatogram. Report the peak area and signal-to-noise ratio (S / N) of each impurity derivative product to be tested in the sensitivity solution; calculate the detection limit concentration of each impurity to be tested and the percentage of this concentration to the concentration of the test solution. The results are shown in Table D. Figure 1 (Chromatogram of sequence number 1) as shown.

[0186] Table D: Sensitivity test results

[0187]

[0188] in conclusion:

[0189] 1 Sensitivity solution was injected three times continuously, and the minimum signal-to-noise ratio of each impurity derivative product peak measured was 23.7, indicating that the quantitative limit concentration and detection limit concentration of chloroacetylhydrazine and trifluoroacetylhydrazine were far less than 0.0298 μg / ml. Therefore, the detection method provided by the present invention has high sensitivity.

[0190] Example 4: Solution stability

[0191] Experimental procedures

[0192] Derivatization reagent: Take 1.0 ml of benzaldehyde and 0.5 ml of formic acid in a 50 ml volumetric flask, dissolve and dilute with acetonitrile, shake well, and obtain;

[0193] Blank solution: Mix 1.0 ml ultrapure water and 1.0 ml derivatization reagent, and react in a 60°C water bath for 2 h to obtain the blank solution.

[0194] Chloroacetylhydrazine stock solution: weigh about 50 mg of chloroacetylhydrazine accurately, put it into a 50 ml volumetric flask, dissolve it with 5.0 ml of 0.1% formic acid water, and then make up to volume with acetonitrile, shake well, and you have it;

[0195] Trifluoroacetylhydrazine stock solution: Weigh about 50 mg of trifluoroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it in acetonitrile and make up to volume, and shake well;

[0196] Reference substance stock solution: Pipette 1.0 ml of chloroacetylhydrazine stock solution and 1.0 ml of trifluoroacetylhydrazine stock solution into a 50 ml volumetric flask, dilute to volume with acetonitrile, and shake well;

[0197] Hydrazine compound solution: Pipette 1.0 ml of reference stock solution into a 100 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0198] Reference solution: Take 1.0 ml of hydrazine compound solution and 1.0 ml of derivatization reagent, mix, and react in a 60°C water bath for 2 hours to obtain the reference solution.

[0199] Test sample spike solution: Take about 20 mg of the test sample, weigh accurately, dissolve it in 1.0 ml of hydrazine compound solution, add 1.0 ml of derivatization reagent, and react in a 60°C water bath for 2 hours to obtain a 100% test sample spike solution;

[0200] Take a needle of the reference solution and the test solution at different times (see Table E for each time point), and calculate the ratio of the peak area of ​​chloroacetylhydrazine derivatives and trifluoroacetylhydrazine derivatives to the peak area at 0h at each time point. The stability results are shown in Table E.

[0201] Table E: Solution Stability Results

[0202]

[0203] in conclusion:

[0204] After the reference solution and the test sample spiked solution were placed at room temperature for 7.5 h and 5.7 h, respectively, the ratios of the areas of chloroacetylhydrazine derivatives and trifluoroacetylhydrazine derivatives to the corresponding derivative product peak areas measured at 0 h were both between 0.85 and 1.24 (the acceptable standard was between 0.70 and 1.30). Therefore, the reference solution and the test sample spiked solution provided by the present invention have good stability.

[0205] Example 5: Detection of chloroacetylhydrazine and trifluoroacetylhydrazine in the starting material of sitagliptin phosphate (3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride) (hereinafter referred to as TTTP)

[0206] (1) Determination of limits for chloroacetylhydrazine and trifluoroacetylhydrazine

[0207] The maximum daily dose of sitagliptin phosphate is 128.5 mg / day. According to ICH Guideline M7, TTC (Threshold of Toxicological Concern) is 1.5 μg / day, and the limit of chloroacetylhydrazine or trifluoroacetylhydrazine is calculated to be 11.67 ppm, which is strictly controlled to 10 ppm.

[0208] (2) Experimental procedures

[0209] Derivatization reagent: Take 1.0 ml of benzaldehyde and 0.5 ml of formic acid in a 50 ml volumetric flask, dissolve and dilute with acetonitrile, shake well, and obtain;

[0210] Blank solution: Mix 1.0 ml ultrapure water and 1.0 ml derivatization reagent, and react in a 60°C water bath for 2 h to obtain the blank solution.

[0211] Chloroacetylhydrazine stock solution: weigh about 50 mg of chloroacetylhydrazine accurately, put it into a 50 ml volumetric flask, dissolve it with 5.0 ml of 0.1% formic acid water, and then make up to volume with acetonitrile, shake well, and you have it;

[0212] Trifluoroacetylhydrazine stock solution: Weigh about 50 mg of trifluoroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it in acetonitrile and make up to volume, and shake well;

[0213] Reference substance stock solution: Pipette 1.0 ml of chloroacetylhydrazine stock solution and 1.0 ml of trifluoroacetylhydrazine stock solution into a 50 ml volumetric flask, dilute to volume with acetonitrile, and shake well;

[0214] Hydrazine compound solution: Pipette 1.0 ml of reference stock solution into a 100 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0215] Reference solution: Take 1.0 ml of hydrazine compound solution and 1.0 ml of derivatization reagent, mix, and react in a 60°C water bath for 2 hours to obtain the reference solution.

[0216] Test solution: weigh about 20 mg of the test sample (sitagliptin phosphate starting material TTTP), add 1.0 ml ultrapure water to dissolve, 1.0 ml of derivatization reagent, and react in a 60°C water bath for 2 h to obtain the solution;

[0217] Take 1 injection of blank solution, 1 injection of detection limit solution, 3 injections of reference solution, and 1 injection of test sample solutions of different batches. Calculate the content of chloroacetylhydrazine and trifluoroacetylhydrazine in the test sample by external standard method. If the content of chloroacetylhydrazine and trifluoroacetylhydrazine in the test sample is less than the detection limit (3.3ppm), the final result is reported as not detected. The results are shown in Table F.

[0218] Table F: Test results of chloroacetylhydrazine and trifluoroacetylhydrazine in starting material TTTP

[0219]

[0220] The test results in Table F show that no chloroacetylhydrazine and trifluoroacetylhydrazine were detected in the seven batches of sitagliptin phosphate starting material TTTP tested.

[0221] Example 6: Screening of derivatization reagents (using benzoyl chloride as the derivatization reagent)

[0222] 1. Solution preparation:

[0223] Derivatization reagent: Take 2.0 ml of benzoyl chloride in a 5 ml volumetric flask, dissolve it in acetonitrile and make up to volume, shake well, and you have it;

[0224] Chloroacetylhydrazine stock solution 1: Weigh about 50 mg of chloroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it with 5.0 ml of 0.1% formic acid water, and then make up to volume with acetonitrile, shake well, and you have it;

[0225] Trifluoroacetylhydrazine stock solution 1: Weigh about 50 mg of trifluoroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it in acetonitrile and make up to volume, and shake well;

[0226] Reference substance stock solution 2: Pipette 1.0 ml of chloroacetylhydrazine stock solution 1 and 1.0 ml of trifluoroacetylhydrazine stock solution 1 into a 50 ml volumetric flask, dilute to volume with acetonitrile, and shake well;

[0227] Reference substance stock solution 3: Pipette 1.0 ml of reference substance stock solution 2 into a 100 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0228] Reference solution: 1.0 ml of reference stock solution 3 and 0.2 ml of derivatization reagent are added to a 10 ml EP tube, and the mixture is reacted at 60 °C for 1 h or 2 h. Take one injection of the reference solution and record the chromatogram. Report the peak area and signal-to-noise ratio of the chloroacetylhydrazine derivative and trifluoroacetylhydrazine derivative products in the reference solution. The results are shown in Table G. After 1 hour, take one injection of the reference solution (equivalent to 2 hours of derivatization reaction) and record the chromatogram. Report the peak area of ​​the chloroacetylhydrazine derivative and trifluoroacetylhydrazine derivative products in the reference solution. The results are shown in Table H.

[0229] 2. Results:

[0230] Table G: Peak area and signal-to-noise ratio of the reference solution derived product after 1 hour of benzoyl chloride derivatization reaction

[0231]

[0232] Table H: Peak area and signal-to-noise ratio of the reference solution after 2 hours of benzoyl chloride derivatization reaction

[0233]

[0234] Conclusion: This indicates that the peak responses of target derivatives are poor when benzoyl chloride is used as the derivatization reagent.

[0235] Example 7: Investigation of mass spectrometry parameters

[0236] 1. Chromatographic and mass spectrometry conditions

[0237] Chromatographic column: Waters HSS T3 column, 4.6 mm × 100 mm, 2.5 μm;

[0238] Detector: Liquid chromatography mass spectrometry detector;

[0239] Ion source: ESI;

[0240] Scan mode: SIM m / z = 197 (+); m / z = 215 (-);

[0241] Flow rate: 1.0ml / min;

[0242] Column temperature: 30°C;

[0243] Injection volume: see Table I;

[0244] Mobile phase A: 5% acetonitrile-95% (0.1% formic acid in water);

[0245] Mobile phase B: acetonitrile;

[0246] Elution ratio:

[0247] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 10 60 40 12 60 40

[0248] Mass spectrometry parameters:

[0249]

[0250] Running time: 12 minutes;

[0251] Post-run: 5min;

[0252] MS acquisition time: 4-12 min.

[0253] 2. Solution preparation:

[0254] Derivatization reagent: Take 1.0 ml of benzaldehyde and add it to a 50 ml volumetric flask, dissolve it with acetonitrile and make up to volume, shake well, and you have it;

[0255] Chloroacetylhydrazine stock solution 1: Weigh about 50 mg of chloroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it with 5.0 ml of 0.1% formic acid water, and then make up to volume with acetonitrile, shake well, and you have it;

[0256] Trifluoroacetylhydrazine stock solution 1: Weigh about 50 mg of trifluoroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it in acetonitrile and make up to volume, and shake well;

[0257] Reference substance stock solution 2: Pipette 1.0 ml of chloroacetylhydrazine stock solution 1 and 1.0 ml of trifluoroacetylhydrazine stock solution 1 into a 50 ml volumetric flask, dilute to volume with acetonitrile, and shake well;

[0258] Reference substance stock solution 3: Pipette 1.0 ml of reference substance stock solution 2 into a 100 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0259] Reference solution: add 1.0ml reference stock solution 3 and 1.0ml derivatization reagent to a 10ml EP tube and react in a 60℃ water bath for 30min.

[0260] Take the reference solution and inject 1 needle, record the chromatogram. Report the peak area of ​​chloroacetylhydrazine derivatives and trifluoroacetylhydrazine derivatives in the reference solution. The results are shown in Table J. Figure 2 (Example 7-1), Figure 3 (Example 7-2), Figure 4 (Example 7-3).

[0261] Table I: Mass spectrometry parameters investigated

[0262]

[0263]

[0264] Table J: Mass spectrometry parameter conditions reference solution derivative product peak area and signal-to-noise ratio results

[0265]

[0266] in conclusion:

[0267] 1. The derivative products using capillary voltages of 3500V(+), 3500(-) and 4500V(+), 4500(-) have high responses and high sensitivity. Among them, using capillary voltages of 3500V(+), 3500(-) is more conducive to improving the response of the derivative products and more conducive to improving the detection sensitivity.

[0268] 2. Using a 90V cleavage voltage is beneficial to improving the response of chloroacetylhydrazine derivatives and trifluoroacetylhydrazine derivatives.

[0269] Example 8: Derivatization time study

[0270] Derivatization reagent: Take 1.0 ml of benzaldehyde and 0.5 ml of formic acid in a 50 ml volumetric flask, dissolve and dilute with acetonitrile, shake well, and obtain;

[0271] Blank solution: Mix 1.0 ml ultrapure water and 1.0 ml derivatization reagent, and react in a 60°C water bath for 1.5 h and 2 h respectively.

[0272] Chloroacetylhydrazine stock solution 1: Weigh about 50 mg of chloroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it with 5.0 ml of 0.1% formic acid water, and then make up to volume with acetonitrile, shake well, and you have it;

[0273] Trifluoroacetylhydrazine stock solution 1: Weigh about 50 mg of trifluoroacetylhydrazine accurately into a 50 ml volumetric flask, dissolve it in acetonitrile and make up to volume, and shake well;

[0274] Reference substance stock solution 2: Pipette 1.0 ml of chloroacetylhydrazine stock solution 1 and 1.0 ml of trifluoroacetylhydrazine stock solution 1 into a 50 ml volumetric flask, dilute to volume with acetonitrile, and shake well;

[0275] Reference substance stock solution 3: Pipette 1.0 ml of reference substance stock solution 2 into a 100 ml volumetric flask, dilute to volume with ultrapure water, and shake well;

[0276] Reference solution: Take 1.0 ml of reference stock solution 3 and 1.0 ml of derivatization reagent, mix, and react in a 60°C water bath for 1.5 h and 2 h respectively.

[0277] Test solution: Take about 20 mg of the test sample, accurately weigh it into a 10 ml EP tube, dissolve it with 1.0 ml ultrapure water, add 1.0 ml of derivatization reagent, and react in a 60 ° C water bath for 1.5 h and 2 h to obtain the test solution;

[0278] Test sample spike solution: Take about 20 mg of the test sample, accurately weigh it into a 10 ml EP tube, dissolve it with 1.0 ml of reference substance stock solution 3, add 1.0 ml of derivatization reagent, and react in a 60 ° C water bath for 1.5 h and 2 h to obtain a 100% test sample spike solution;

[0279] Take each of the above solutions according to the chromatographic conditions described in the method, inject one needle into each portion, and record the chromatogram. Report the retention time, peak area and separation degree of each impurity derivative product to be tested in the blank solution, reference solution, test solution, and test solution spiked with the test product;

[0280] Calculate the spike recovery (R value), the calculation formula of R value is as follows:

[0281]

[0282] Where: A T+S is the peak area of ​​each impurity-derived product in the test sample spiked solution;

[0283] A T is the peak area of ​​each impurity-derived product in the test solution;

[0284] A S It is the average of the peak areas of each impurity derivative product in 3 reference solutions;

[0285] W T is the weight of the test sample in the test solution, mg;

[0286] W T+S is the weight of the test sample in the test sample spike solution, mg.

[0287] Results: See Table K.

[0288] Table K: Derivatization time investigation results

[0289]

[0290] Conclusion: A derivatization time of 1.5h-2h can obtain results with good separation, high response (i.e. high sensitivity) and high accuracy.

[0291] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for detecting hydrazine compounds, characterized in that, include: The sample to be tested dissolved in water is mixed with a derivatization reagent to cause a derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent to produce a derivative product, wherein the derivatization reagent includes benzaldehyde, and then the content of the derivative product is detected by high performance liquid chromatography-mass spectrometry, and then the content of the hydrazine compound is calculated.

2. The detection method according to claim 1, wherein the hydrazine compound is a hydrazide compound, and / or The hydrazide compound includes at least one of chloroacetohydrazide or trifluoroacetohydrazide.

3. The detection method according to any one of claims 1 to 2, wherein the derivatization reagent comprises benzaldehyde; and / or The derivatization reagent comprises benzaldehyde and an acid; and / or The derivatization reagent is a solution containing benzaldehyde, acid and solvent; and / or The acid comprises at least one of formic acid, acetic acid and phosphoric acid; and / or The solvent comprises at least one of acetonitrile, methanol and water; and / or The concentration of benzaldehyde in the derivatization reagent is 1.0% vol-4.0% vol or 2.0% vol; and / or The concentration of the acid in the derivatization reagent is 0.5% vol-2.0% vol or 1.0% vol.

4. The detection method according to claim 3, wherein 10 mg to 30 mg of the sample to be tested is added per 1 ml of the derivatization reagent; or 20 mg of the sample to be tested is added per 1 ml of the derivatization reagent; and / or Dissolve 5mg-50mg of the sample to be tested per 1ml of water; or dissolve 10mg-30mg of the sample to be tested per 1ml of water; or dissolve 15mg-25mg of the sample to be tested per 1ml of water; or dissolve 20mg of the sample to be tested per 1ml of water; and / or The reaction temperature of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent is 50-65° C. or 60° C.; and / or The reaction time of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent is 1.5-2.5h or 2h.

5. The detection method according to any one of claims 1 to 4, characterized in that: The method also includes the preparation and detection of a reference solution, wherein the preparation of the reference solution includes: mixing a hydrazine compound solution with a derivatization reagent to generate a derivatization reaction to obtain the reference solution; or The concentration of the hydrazine compound in the hydrazine compound solution is 0.1 μg / ml-0.5 μg / ml or 0.2 μg / ml-0.4 μg / ml; and / or The reaction temperature of the derivatization reaction in the preparation of the reference solution is 50°C-65°C or 60°C; and / or The reaction time of the derivatization reaction in the preparation of the reference solution is 1.5h-2.5h or 2h; and / or In the preparation of the reference solution, the volume ratio of the hydrazine compound solution to the derivatization reagent is 1:1-1:2 or 1:1; and / or The reaction temperature and reaction time of the derivatization reaction between the hydrazine compound in the sample to be tested and the derivatization reagent are respectively the same as the reaction temperature and reaction time of the derivatization reaction in the preparation method of the reference solution; and / or The hydrazine compound includes chloroacetylhydrazine and trifluoroacetylhydrazine, and the preparation of the reference solution includes: mixing a hydrazine compound solution containing 0.1 μg / ml-0.5 μg / ml chloroacetylhydrazine and 0.1 μg / ml-0.5 μg / ml trifluoroacetylhydrazine with a derivatization reagent, reacting at 50-65° C. for 2.0 hours to obtain the reference solution, wherein the concentration is a volume ratio of the hydrazine compound solution to the derivatization reagent of 1:1-1:2; or The hydrazine compound includes chloroacetylhydrazine and trifluoroacetylhydrazine, and the preparation of the reference solution includes: mixing a hydrazine compound solution containing 0.2 μg / ml chloroacetylhydrazine and 0.2 μg / ml trifluoroacetylhydrazine with a derivatization reagent, reacting at 60° C. for 2 hours to obtain the reference solution, wherein the concentration is a volume ratio of the hydrazine compound solution to the derivatization reagent of 1:1; and / or The solvent of the hydrazine compound solution is an organic solvent and / or water; and / or The preparation method of the hydrazine compound solution comprises: dissolving chloroacetylhydrazine with 0.05%vol-0.20%vol or 0.10%vol acid aqueous solution and then diluting with an organic solvent to obtain a chloroacetylhydrazine stock solution; diluting trifluoroacetylhydrazine with an organic solvent to obtain a trifluoroacetylhydrazine stock solution; mixing the chloroacetylhydrazine stock solution and the trifluoroacetylhydrazine stock solution, and diluting with an organic solvent to obtain a reference substance stock solution, and then diluting the reference substance stock solution with water to obtain the hydrazine compound solution; and / or The acid in the aqueous acid solution is formic acid; and / or The concentration of chloroacetylhydrazine in the chloroacetylhydrazine stock solution is 0.5mg / ml-2.5mg / ml or 1.0mg / ml; and / or The concentration of trifluoroacetylhydrazine in the trifluoroacetylhydrazine stock solution is 0.5 mg / ml-2.5 mg / ml or 1.0 mg / ml; and / or The concentration of chloroacetylhydrazine in the reference substance stock solution is 0.01 mg / ml-0.05 mg / ml or 0.02 mg / ml; and / or The concentration of trifluoroacetylhydrazine in the reference substance stock solution is 0.01 mg / ml-0.05 mg / ml or 0.02 mg / ml; and / or The organic solvent is acetonitrile.

6. The detection method according to any one of claims 1 to 5, wherein the high performance liquid chromatography is performed using a C18 chromatographic column, preferably a Waters HSS T3 columns; and / or Waters The length of HSS T3 column is 100mm-250mm; and / or Waters The inner diameter of the HSS T3 column is 2mm-6mm, 3mm-5mm or 3.9mm-4.6mm; and / or Waters The particle size of the filler of the HSS T3 column is 1.8 μm-5 μm, 2 μm-3 μm or 2.5 μm; and / or The high performance liquid chromatography method uses a mixture of acetonitrile and 0.05% vol-0.20% vol formic acid aqueous solution in a volume ratio of 2.5:97.5-10.0:90.0 as mobile phase A, acetonitrile as mobile phase B, and performs isocratic elution or gradient elution; or the high performance liquid chromatography method uses a mixture of acetonitrile and 0.10% vol formic acid aqueous solution in a volume ratio of 5.0:95.0 as mobile phase A, acetonitrile as mobile phase B, and performs isocratic elution or gradient elution; and / or The isocratic elution is performed by using a volume ratio of mobile phase A to mobile phase B of 75:25 to 85:15, 78:22 to 82:18 or 80:20; and / or The isocratic elution time is 12 min to 25 min; and / or The elution procedure of the gradient elution is: ； Or the elution program of the gradient elution is: ； Or the elution program of the gradient elution is: ； Or the elution program of the gradient elution is: 。 7. The detection method according to any one of claims 1 to 6, wherein the flow rate of the HPLC is 0.8 ml / min-1.2 ml / min; or the flow rate of the HPLC is 0.9 ml / min-1.1 ml / min; or the flow rate of the HPLC is 1.0 ml / min; and / or The column temperature of the HPLC is 15°C-40°C; or the column temperature of the HPLC is 25°C-35°C; or the column temperature of the HPLC is 28°C-32°C; or the column temperature of the HPLC is 29°C-31°C; or the column temperature of the HPLC is 30°C; and / or The injection volume of the high performance liquid chromatography is 5 μL-50 μL or 10 μL-20 μL; and / or The elution program of the HPLC method also includes a post-run time of 2-10 min.

8. The detection method according to any one of claims 1 to 7, The ion source of the mass spectrometry is an electrospray ion source; and / or The mass spectrometry scanning mode is SIM m / z=197(+); m / z=215(-); and / or The mass spectrometry acquisition time is 4 min-25 min, 4 min-20 min, 4 min-15 min or 4 min-12 min; and / or The drying gas temperature of the mass spectrometry is 300°C-350°C or 300°C; and / or The mass spectrometry drying gas flow rate is 8 L / min-12 L / min or 12 L / min; and / or The mass spectrometry nebulizer gas pressure is 30psi-50psi or 45psi; and / or The capillary voltage of the mass spectrometry is 3500 V (+), 3500 V (-), or the capillary voltage of the mass spectrometry is 4500 V (+), 4500 V (-); The fragmentation voltage of the mass spectrometry method is 70V-90V.

9. The detection method according to claim 1, comprising: The sample to be tested is dissolved in water and then mixed with a derivatization reagent, so that the hydrazine compound in the sample to be tested reacts with the derivatization reagent at 60°C for 2 hours to produce a derivative product, wherein the derivatization reagent is an acetonitrile solution containing 2.0% vol benzaldehyde and 1.0% vol formic acid; then, the content of the derivative product is detected by high performance liquid chromatography and mass spectrometry, and the content of the hydrazine compound is calculated; the high performance liquid chromatography adopts Waters HSS T3 column was used for detection. The HSS T3 chromatographic column has a column length of 100 mm, an inner diameter of 4.6 mm, and a filler particle size of 2.5 μm. The high performance liquid chromatography method uses a mixture of acetonitrile and 0.10% vol formic acid aqueous solution in a volume ratio of 5.0:95.0 as mobile phase A, and acetonitrile as mobile phase B for gradient elution. The elution procedure of the gradient elution is: ； The flow rate of the HPLC method is 1.0 ml / min; The column temperature of the HPLC method is 30°C; The elution procedure of the high performance liquid chromatography also includes a post-run time of 5 min; The mass spectrometry method adopts a liquid chromatography-mass spectrometer; the ion source of the mass spectrometry method is an electrospray ion source; the scanning mode of the mass spectrometry method is SIM m / z=197(+); m / z=215(-); the acquisition time of the mass spectrometry method is 4min-12min; the drying gas temperature of the mass spectrometry method is 300°C; the drying gas flow rate of the mass spectrometry method is 12L / min; the nebulizing gas pressure of the mass spectrometry method is 45psi; the capillary voltage of the mass spectrometry method is 3500V(+), 3500V(-), or the capillary voltage of the mass spectrometry method is 4500V(+), 4500V(-); the fragmentation voltage of the mass spectrometry method is 70V-90V.

10. Use of the detection method according to any one of claims 1 to 9 in detecting the content of chloroacetylhydrazine and / or trifluoroacetylhydrazine in sitagliptin or its salt, a preparation of sitagliptin or its salt, (3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine or its salt, a raw material for synthesizing sitagliptin or its salt, or an intermediate compound for synthesizing sitagliptin or its salt.

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