CN109655539B - Detection method of ethylenediamine and method for detecting ethylenediamine in sitagliptin phosphate intermediate XG-SM2 - Google Patents

Abstract

The invention provides an ethylenediamine detection method based on ion chromatography, and the chromatographic conditions are as follows: a chromatographic column: thermo Dionex ion pac CS12A cation chromatography column, 250mm 4 mm; a detector: a conductivity detector; mobile phase: 40mmol/l methanesulfonic acid; flow rate: 0.7ml/min-1.4 ml/min; column temperature: 28-35 ℃; sample introduction amount: 25 ul. The lowest linear detection concentration of the detection method is 0.4 mu g/ml, and the lowest detection concentration of the detection method is 0.06 mu g/ml determined by a signal-to-noise ratio of 3: 1. The invention also provides application of the detection method in impurity detection of the sitagliptin phosphate intermediate XG-SM 2.

Description

Detection method of ethylenediamine and method for detecting ethylenediamine in sitagliptin phosphate intermediate XG-SM2

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a detection method of ethylenediamine and a method for detecting ethylenediamine in a sitagliptin phosphate intermediate XG-SM 2.

Background

Diabetes mellitus is a chronic complex disease mainly caused by glucose metabolism disorder, and comprises insulin-dependent diabetes mellitus (type I diabetes mellitus), non-insulin-dependent diabetes mellitus (type II diabetes mellitus, 90 percent) and gestational diabetes mellitus. The global diabetes map (8 th edition) released by the international diabetes consortium shows that the global diabetes prevalence (20-79 years) in 2017 is about 8.8%, and about 4.25 million adult diabetics, and by 2045 years, the number may reach 6.29 million.

China is the country with the largest number of diabetics, and accounts for about one fourth of the world. Diabetes, as a high-incidence chronic disease, has the characteristics of irreversible disease condition, more late complications and the like. If the blood sugar is not well controlled, diabetic nephropathy, diabetic retinopathy and other complications can be caused by the chronicity of the diabetes, so that renal failure, blindness and other serious consequences can be caused, and huge burden is caused to the society and families. Meanwhile, diabetes is also a risk factor causing a plurality of cardiovascular diseases, such as myocardial infarction, cerebral apoplexy and the like.

The research of related blood sugar reducing mechanism shows that when blood sugar in a human body is increased, the secretion of active intestinal insulinotropic hormone glucagon-like peptide-1 (GLP-l) and glucose-dependent insulinotropic secretion peptide (GIP) can be promoted, so that the synthetic secretion process of insulin is further stimulated, and the effect of controlling blood sugar is finally achieved. GLP-1 and GIP in vivo are easily degraded and inactivated by dipeptidyl peptidase IV (DPP-IV); thus, dipeptidyl peptidase IV is a target for the treatment of diabetes. Sitagliptin phosphate is the first dipeptidyl peptidase-4 inhibitor (DPP-4 inhibitor) approved by the United states Food and Drug Administration (FDA) for treating type II diabetes, can effectively inhibit the inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), improve the levels of endogenous GLP-1 and GIP, promote insulin release of islet beta cells, and inhibit glucagon secretion of islet alpha cells, so that insulin levels are improved, blood sugar is reduced, hypoglycemia is not easily induced, and weight is increased.

Sitagliptin phosphate, chemical name: 7- [ (3R) -3-amino-1-oxo-4- (2,4, 5-trifluorophenyl) butyl]-5,6,7, 8-tetrahydro-3- (trifluoromethyl) -1,2, 4-triazolone [4,3-a ]]Pyrazine phosphate (1:1) monohydrate, chemical formula C₁₆H₁₅F₆N₅O·H₃PO₄·H₂O, the chemical structure is shown as formula (I).

3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine hydrochloride (hereinafter referred to as 'XG-SM 2') with a structural formula shown as formula (II) is an important intermediate in the process of synthesizing sitagliptin phosphate. Ethylenediamine was the starting material for the synthesis of this intermediate. If the synthesis reaction is insufficient, ethylenediamine impurities tend to remain. Under the condition that the safety of the medicines is increasingly emphasized, the detection of the trace impurities remained in the medicines is gradually brought into the quality standard of the medicines or the internal control standard of enterprises. Therefore, it is necessary to establish a method for detecting ethylenediamine in 3- (trifluoromethyl) -5,6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine hydrochloride so as to ensure the safety of clinical patients.

The ethylene diamine has larger molecular polarity, and extremely poor retention in a common silica gel adsorption column; and the structure has no chromophoric group and no ultraviolet absorption; therefore, the method cannot be separated by using a liquid chromatograph using common silica gel as a stationary phase and cannot be used by using an ultraviolet detector. In this regard, various solutions have emerged in the prior art. For example, the Chinese patent application with publication number CN106596795A discloses that the content of ethylenediamine in the injection of thioctic acid is determined by using a Waters Xbridge Amide chromatographic column (Amide bonded stationary phase) for separation and combining a refractive index detector. Also, as disclosed in the chinese patent application publication No. CN106053698A, ethylene diamine is derivatized with p-benzenesulfonyl chloride, then separated with a silica gel C18 column, and the derivative is measured with an ultraviolet detector.

The ion chromatography is a chromatographic method which takes ion exchange resin or chemical bonding ion exchanger as a stationary phase and realizes separation by utilizing the difference of ion exchange capacity or selectivity coefficient of separated components, and is a special high performance liquid chromatography. There have been many reports in the prior art for detecting polar amine compounds using ion chromatography. For example, the Chinese patent application with publication number CN104215723A discloses a method for measuring the contents of monomethylamine and monoethylamine in cigarette flow measurement smoke by using ion chromatography, wherein the chromatographic columns are a CS16 separation column and a CG16 protection column, the mobile phase is 10mmol/L of methane sulfonic acid, and isocratic elution is carried out. Further, as disclosed in the chinese patent application publication No. CN103454367A, a method for detecting monomethylamine by ion chromatography is also disclosed, wherein a chromatographic column is IonPac CS12A, and a mobile phase is 16mmol/L methanesulfonic acid.

So far, no report of detecting ethylenediamine in the sitagliptin phosphate intermediate XG-SM2 by using ion chromatography is found.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for detecting ethylenediamine by using ion chromatography, and the method is applied to the qualitative and/or quantitative detection of ethylenediamine in the sitagliptin phosphate intermediate XG-SM 2. According to the method, the peak emergence time of the ethylenediamine is about 10min, the lowest linear detection concentration is 0.4 mu g/ml, and the lowest detection concentration of the method is 0.06 mu g/ml determined by a signal-to-noise ratio of 3: 1; and the sample adding and recovering experiment shows that other components in the test solution do not interfere with the detection of the ethylenediamine. The method can realize the purpose of quickly and effectively detecting the content of the ethylenediamine in the sitagliptin phosphate intermediate XG-SM2, and fills the blank in the prior art.

To achieve the above object of the invention. The invention adopts the following technical scheme:

a method for detecting ethylenediamine, said method based on ion chromatography, comprising:

establishment of chromatographic conditions:

a chromatographic column: thermo Dionex ion pac CS12A cation chromatography column, 250mm 4 mm;

protection of the column: thermo Dionex ion pac CG12 guard column, 4mm x 50 mm;

a detector: a conductance detector;

mobile phase: 40mmol/l methanesulfonic acid;

flow rate: 0.7ml/min-1.4 ml/min;

column temperature: 28-35 ℃;

sample introduction amount: 25 ul.

The flow rate is preferably 1.0 ml/min.

The column temperature is preferably 32 ℃.

The detection method also comprises the preparation of a reference substance solution, and the specific steps comprise:

precisely measuring a proper amount of ethylenediamine, dissolving with ultrapure water, and diluting into a solution containing 2 mu g of ethylenediamine per 1 ml.

In the detection method, the preparation of the test solution also adopts ultrapure water as a solvent.

The detection method also comprises qualitative detection and/or quantitative determination, and the specific steps comprise:

and (3) qualitative detection:

under the chromatographic condition, precisely measuring 25 mu l of reference solution, injecting the reference solution into an ion chromatograph, and adjusting the sensitivity of the detector to ensure that the chromatographic peak height of the main component is 10-20% of the full-scale range; precisely measuring 25 μ l of each of the test solution and the reference solution, injecting sample, injecting into ion chromatograph, and recording chromatogram; comparing the chromatogram with the chromatogram of the reference solution, and observing whether a corresponding chromatographic peak appears in the chromatogram of the test solution at a corresponding retention time;

and/or

Quantitative determination:

under the chromatographic condition, precisely measuring 25 mu l of reference solution, injecting the reference solution into an ion chromatograph, and adjusting the sensitivity of the detector to ensure that the chromatographic peak height of the main component is 10-20% of the full-scale range; and precisely measuring 25 mul of each of the test solution and the reference solution, injecting the samples respectively, injecting the samples into an ion chromatograph, and calculating the content of the ethylenediamine in the test solution according to an external standard method.

The detection method of the invention has the lowest linear detection concentration of 0.4 mug/ml and the lowest detection concentration of 0.06 mug/ml determined by the signal-to-noise ratio of 3: 1.

The invention also aims to provide the application of the detection method in the impurity detection of the sitagliptin phosphate intermediate XG-SM2 with the structural formula shown in the formula (II),

specifically, the application is that the detection method provided by the invention is utilized to carry out qualitative detection and/or quantitative determination on the ethylenediamine in the sitagliptin phosphate intermediate XG-SM 2.

The invention also provides a method for detecting ethylenediamine in the sitagliptin phosphate intermediate XG-SM2 with the structural formula shown in the formula (II),

comprises the establishment of chromatographic conditions, the preparation of reference solution, the preparation of test solution, qualitative detection and/or quantitative determination, wherein,

establishment of chromatographic conditions:

a chromatographic column: thermo Dionex ion pac CS12A cation chromatography column, 250mm 4 mm;

protection of the column: thermo Dionex ion pac CG12 guard column, 4mm x 50 mm;

a detector: a conductance detector;

mobile phase: 40mmol/l methanesulfonic acid;

flow rate: 0.7ml/min-1.4 ml/min; preferably 1.0 ml/min;

column temperature: 28-35 ℃; preferably 32 ℃;

sample introduction amount: 25 ul;

preparation of control solutions:

precisely measuring a proper amount of ethylenediamine, dissolving with ultrapure water, and diluting into a solution containing 2 mu g of ethylenediamine per 1ml to obtain the ethylenediamine aqueous solution;

preparation of a test solution:

taking a proper amount of the sitagliptin phosphate intermediate XG-SM2, adding ultrapure water for dissolving, and diluting into a solution containing 1mg of the sitagliptin phosphate intermediate XG-SM2 per 1ml to obtain the sitagliptin phosphate intermediate XG-SM 2;

and (3) qualitative detection:

under the chromatographic condition, precisely measuring 25 mu l of reference solution, injecting the reference solution into an ion chromatograph, and adjusting the sensitivity of the detector to ensure that the chromatographic peak height of the main component is 10-20% of the full-scale range; precisely measuring 25 μ l of each of the test solution and the reference solution, injecting sample, injecting into ion chromatograph, and recording chromatogram; comparing the chromatogram with the chromatogram of the reference solution, and observing whether a corresponding chromatographic peak appears in the chromatogram of the test solution at a corresponding retention time;

and/or

Quantitative determination:

under the chromatographic condition, precisely measuring 25 mu l of reference solution, injecting the reference solution into an ion chromatograph, and adjusting the sensitivity of the detector to ensure that the chromatographic peak height of the main component is 10-20% of the full-scale range; and precisely measuring 25 mul of each of the test solution and the reference solution, injecting the samples respectively, injecting the samples into an ion chromatograph, and calculating the content of the ethylenediamine in the test solution according to an external standard method.

According to the method for determining the ethylenediamine in the sitagliptin phosphate intermediate XG-SM2, which is preferably established, the treatment such as sample derivatization is not needed, and the pretreatment of the sample to be determined is simple. The time for completing the detection of one sample is short, and the operability is strong. The method is strong in specificity, and other components in the sitagliptin phosphate intermediate XG-SM2 do not interfere with detection of ethylenediamine. The method has high sensitivity, the lowest linear detection concentration is 0.4 mu g/ml, and the lowest detection concentration determined by the signal-to-noise ratio of 3:1 is 0.06 mu g/ml. Therefore, the detection method of the ethylenediamine provided by the invention can be used for effectively and quickly detecting the ethylenediamine in the sitagliptin phosphate intermediate XG-SM2, so that the quality of products is better controlled, and the safety use of medicines is guaranteed.

Drawings

The present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 shows the chromatogram of the ethylenediamine control from gradient elution with a mobile phase of 15mmol/L → 50mmol/L methanesulfonic acid and a flow rate of 1.0ml/min in example 1; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 2 shows the chromatogram of the ethylenediamine control from gradient elution with a mobile phase of 10mmol/L → 40mmol/L methanesulfonic acid and a flow rate of 1.0ml/min in example 1; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 3 shows the chromatogram of the ethylenediamine control from gradient elution with a mobile phase of 20mmol/L → 40mmol/L methanesulfonic acid and a flow rate of 1.0ml/min in example 1; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 4 shows the chromatogram of the ethylenediamine control in example 1 with 16mmol/L methanesulfonic acid as the mobile phase, a flow rate of 1.0ml/min, and isocratic elution; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 5 shows the chromatogram of the ethylenediamine control in example 1 with a mobile phase of 35mmol/L methanesulfonic acid, a flow rate of 1.0ml/min, and isocratic elution; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 6 shows the chromatogram of the ethylenediamine control in example 1 with a mobile phase of 40mmol/L methanesulfonic acid, a flow rate of 1.0ml/min, and isocratic elution; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 7 shows a chromatogram of an ethylenediamine control in example 1 with a mobile phase of 45mmol/L methanesulfonic acid, a flow rate of 1.0ml/min, and isocratic elution; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 8 shows a chromatogram of an ethylenediamine control of example 1 with a flow rate of 0.7ml/min and a mobile phase of 40mmol/L methanesulfonic acid; in the figure, the peak labeled 1 is the peak of ethylenediamine.

FIG. 9 shows a chromatogram of an ethylenediamine control of example 1 with a flow rate of 1.4ml/min and a mobile phase of 40mmol/L methanesulfonic acid; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 10 shows a chromatogram of an ethylene diamine control solution from the proprietary test of example 2; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 11 is a chromatogram of a test solution in the proprietary test of example 2; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 12 shows the regression curve of the control solution in the linear range test of example 2, wherein the abscissa (x) is the ethylenediamine concentration and the ordinate (y) is the chromatographic peak area.

FIG. 13 shows a chromatogram of a control solution (ethylenediamine concentration 0.06. mu.g/ml) in the detection limit test in example 2; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

FIG. 14 is a chromatogram of a sample solution numbered 100% -1 in the sample recovery test in example 2; in the figure, the chromatographic peak designated as 1 is the chromatographic peak of ethylenediamine.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified. Wherein, some reagents and instruments are purchased as follows:

ion chromatography: thermo Dionex AQ ion chromatograph, seymel feishell science ltd;

chromatographic column, Thermo Dionex IonPac CS12A cation column (4 mm. about.250 mm), Sammer Feishell technologies Inc.;

protection of the column: thermo Dionex ion pac CG12 guard column (4mm x 50mm), seimer feishell technologies ltd;

an electronic balance: CPA225D sydows scientific instruments (beijing) ltd;

leacheate (methanesulfonic acid solution): EGC III MSA, Sammer Feishel technologies, Inc.;

reagents and reagents:

hydrazine sulfate: sigma aldrich;

ultrapure water: hangzhou child-Ha group;

sitagliptin phosphate intermediate XG-SM 2: tonghua Dongbao pharmaceuticals Co., Ltd, lot Nos. C182016001 and C182017013.

Example 1: selection of mobile phase

The purpose of this example was to examine the effect of concentration and elution program on the separation and detection of ethylenediamine using methanesulfonic acid as the mobile phase.

In addition to the mobile phase, the following chromatographic conditions were employed in the present examples

An ion chromatograph: thermo Dionex Aquion type ion chromatograph;

a chromatographic column: thermo Dionex ion pac CS12A cation column (4mm 250 mm);

protection of the column: thermo Dionex ion pac CG12 guard column (4mm 50 mm);

flow rate: 1.0 ml/min;

column temperature: 28-35 ℃;

a detector: conductivity detector

Sample introduction amount: 25 μ l.

In this example, an ethylenediamine reference solution was used for the measurement, and the preparation method of the ethylenediamine reference solution was:

precisely measuring a proper amount of ethylenediamine, dissolving with ultrapure water, and diluting into a solution containing about 2 μ g of ethylenediamine per 1 ml.

The determination method comprises the following steps:

precisely measuring 25 μ l of the reference solution, injecting into ion chromatograph, and recording chromatogram.

1.1 methanesulfonic acid concentration and preference for elution procedure

(1) Gradient elution of 15mmol/L → 50mmol/L methanesulfonic acid

Elution and measurement were performed according to the gradient elution procedure shown in Table 1, and the chromatogram is shown in FIG. 1.

Table 1 gradient elution procedure 1

Time (min)	Methanesulfonic acid (mmol/L)
		0.0	15
20.0	15
		20.1	50
110.0	50
		110.1	15
140.0	15

As can be seen from fig. 1: under the above elution conditions, the running method for completing the measurement of one sample is long, and ethylenediamine peaks at about 25 minutes (retention time 25.69min), and at the same time, a large gradient peak appears at 110 minutes.

(2) Gradient elution of 10mmol/L → 40mmol/L methanesulfonic acid

Elution and measurement were performed according to the gradient elution procedure shown in Table 2, and the chromatogram is shown in FIG. 2.

Table 2 gradient elution procedure 2

Time (min)	Methanesulfonic acid (mmol/L)
		0.0	10
20.0	10
		20.1	40
40.0	40
		40.1	10
60.0	10

As can be seen from fig. 2: after the mobile phase was adjusted, ethylenediamine peaked at 28.83 minutes, while a larger gradient peak also appeared at 45 minutes.

(3) Gradient elution of 20mmol/L → 40mmol/L methanesulfonic acid

Elution and measurement were performed according to the gradient elution procedure shown in Table 3, and the chromatogram is shown in FIG. 3.

Table 3 gradient elution procedure 3

Time (min)	Methanesulfonic acid (mmol/L)
		0.0	20
10.0	20
		30.0	40
40.0	40
		40.1	20
60.0	20

As can be seen from fig. 3: under the elution condition, the ethylenediamine peaks at about 24 minutes (retention time 23.90 min); at the same time, a distinct gradient peak also appeared at 45 minutes.

None of the three gradient elution conditions described above is suitable for the isolation of ethylenediamine. Therefore, it was decided to use isocratic elution to avoid gradient peaks and to have a suitable retention time for ethylenediamine (around 10 minutes).

(4) Isocratic elution with 16mmol/L methanesulfonic acid

Elution and measurement were carried out using 16mmol/L methanesulfonic acid as the mobile phase, and the chromatogram is shown in FIG. 4.

As can be seen from FIG. 4, after isocratic elution with 16mmol/L, ethylenediamine peaked around 47 minutes (retention time 47.067min) with a ragged baseline.

(5) Isocratic elution with 35mmol/L methanesulfonic acid

Elution and measurement were carried out using 35mmol/L methanesulfonic acid as the mobile phase, and the chromatogram is shown in FIG. 5.

As can be seen from FIG. 5, the peak of ethylenediamine appeared earlier to 10.521 minutes after elution with 35mmol/L methanesulfonic acid, but the peak of ethylenediamine chromatogram was more severely smeared with an uneven baseline.

(6) Isocratic elution with 40mmol/L methanesulfonic acid

Elution and measurement were carried out using 40mmol/L methanesulfonic acid as the mobile phase, and the chromatogram is shown in FIG. 6.

As can be seen from FIG. 6, 40mmol/L methanesulfonic acid is the mobile phase, the peak-off time of ethylenediamine is appropriate (retention time 10.16min), and the baseline is relatively flat.

(7) Isocratic elution with 45mmol/L methanesulfonic acid

Elution and measurement were carried out using 45mmol/L methanesulfonic acid as the mobile phase, and the chromatogram is shown in FIG. 7.

As can be seen from FIG. 7, the ethylenediamine peak-off time was earlier (retention time 7.410min) and the ethylenediamine chromatographic peak still had a more severe tail after using 45mmol/L methanesulfonic acid as the mobile phase.

From the elution effects of the different mobile phases, 40mmol/L methanesulfonic acid is taken as the mobile phase, the retention time of ethylenediamine is appropriate, the absorption peak type is good, no tailing exists basically, and the base line is smooth. Therefore, the preferred mobile phase is 40mmol/L methanesulfonic acid.

The flow rate of the mobile phase also has an influence on the separation effect, and therefore the following experiment was performed with 40mmol/L methanesulfonic acid as the mobile phase to preferably select the optimum flow rate of the mobile phase.

1.2 optimization of the flow Rate of the Mobile phase

(1) Flow rate 0.7ml/min

Elution and measurement were carried out at a flow rate of 0.7ml/min using 40mmol/L methanesulfonic acid as the mobile phase, and the chromatogram was shown in FIG. 8.

As can be seen from FIG. 8, the retention time of ethylenediamine was extended to about 13 minutes (retention time 13.047min) at a flow rate of 0.7ml/min, as compared to a flow rate of 1.0ml/min, and a slight tailing condition occurred.

(2) Flow rate 1.4ml/min

Elution and measurement were carried out at a flow rate of 1.4ml/min using 40mmol/L methanesulfonic acid as the mobile phase, and the chromatogram was shown in FIG. 9.

As can be seen from FIG. 9, when the flow rate was adjusted to 1.4ml/min, the ethylene diamine peak time was advanced to about 6.2 minutes (retention time 6.237min), and the baseline was worse than the flow rate of 1.0 mi/min.

The above test results show that 40mmol/L methanesulfonic acid is used as the mobile phase, the response of the chromatographic peak of ethylenediamine is good when the flow rate is 0.7-1.4ml/min, and particularly when the flow rate is 1.0ml/min, the retention time of ethylenediamine is optimum, the chromatographic peak is good in type, no tailing exists, and the baseline is smooth. Thus, a preferred flow rate of 40mmol/L methanesulfonic acid is 1.0 ml/min.

Example 2Methodology study of the detection method of ethylenediamine of the present invention

On the basis of the embodiment 1, a detection method of ethylenediamine is established, and specifically comprises the following steps:

I. chromatographic conditions are as follows:

ion chromatography: thermo Dionex Aquion type ion chromatograph;

a chromatographic column: thermo Dionex ion pac CS12A cation chromatography column (4mm 250 mm);

protection of the column: thermo Dionex ion pac CG12 guard column (4mm 50 mm);

mobile phase: 40mmol/L methanesulfonic acid (made up in ultrapure water)

Flow rate: 0.7-1.4ml/min, preferably 1.0 ml/min;

column temperature: 28-35 ℃;

a detector: conductivity detector

Sample introduction amount: 25 μ l.

Preparation of control solutions:

precisely measuring a proper amount of ethylenediamine, dissolving with ultrapure water, and diluting into a solution containing about 2 mug of ethylenediamine per 1 ml;

preparation of a test solution:

and taking a proper amount of the sitagliptin phosphate intermediate XG-SM2, adding ultrapure water for dissolving, and diluting into a solution containing about 1mg of the sitagliptin phosphate intermediate XG-SM2 per 1ml, thus obtaining the sitagliptin phosphate intermediate XG-SM 2.

Qualitative detection:

under the chromatographic condition, precisely measuring 25 mu l of reference solution, injecting the reference solution into an ion chromatograph, and adjusting the sensitivity of the detector to ensure that the chromatographic peak height of the main component is 10-20% of the full-scale range; precisely measuring 25 μ l of each of the test solution and the reference solution, injecting sample, injecting into ion chromatograph, and recording chromatogram; and comparing the chromatogram with the chromatogram of the reference solution, and observing whether a corresponding chromatographic peak appears in the chromatogram of the test solution at a corresponding retention time.

Quantitative determination:

under the chromatographic condition, precisely measuring 25 mu l of reference solution, injecting the reference solution into an ion chromatograph, and adjusting the sensitivity of the detector to ensure that the chromatographic peak height of the main component is 10-20% of the full-scale range; and precisely measuring 25 mul of each of the test solution and the reference solution, injecting the samples respectively, injecting the samples into an ion chromatograph, and calculating the content of the ethylenediamine in the test solution according to an external standard method.

In this example, the established detection method for ethylenediamine is validated in terms of specificity, stability, precision, linear detection range, minimum detection limit, durability, accuracy (sample recovery method), and the like.

2.1 specificity

Precisely measuring 25 μ l of each of the sample solution and the reference solution, respectively injecting into an ion chromatograph, and recording chromatogram. The results are shown in Table 4 and FIGS. 10 and 11. .

TABLE 4 results of the specificity test

Specificity experiments	Sample weighing (mg)	Concentration (μ g/ml)	Retention time (min)	Peak area	Number of theoretical plate	Signal to noise ratio
							Ethylene diamine	98.24	1.96	10.16	0.096	3125	232.5
XG-SM2	10.09	1009.00	10.26	0.037	2822	75.5

Test results show that the method can effectively detect the ethylenediamine in the XG-SM2, other components in the test solution do not interfere with the ethylenediamine, and the method has strong specificity.

2.2 stability

Accurately weighing 99.73mg ethylenediamine, preparing stock solution with concentration of 9.97 μ g/ml with ultrapure water, and diluting the stock solution with ultrapure water to obtain 1.99 μ g/ml solution to obtain reference solution.

Precisely weighing 9.96mgXG-SM2, and preparing into solution with concentration of 996.00 mug/ml with ultrapure water to obtain the test solution.

After the control solution and the test solution were prepared, they were left at room temperature (18-23 ℃) and measured for 0h, 4h, 9h, 12h and 24h, respectively, and the results are shown in Table 5.

TABLE 5 stability test results

Hydrazine sulfate	Retention time (min)	Peak area	XG-SM2	Retention time (min)	Peak area
						0h	10.05	0.111	0h	10.12	0.035
4h	10.04	0.111	4h	10.11	0.035
						9h	10.09	0.111	9h	10.14	0.036
12h	10.07	0.110	12h	10.09	0.036
						24h	10.01	0.109	24h	10.05	0.035
Mean value of	10.05	0.110	Mean value of	10.10	0.040
						RSD％	0.30％	0.81％	RSD％	0.34％	1.37％

The data in Table 5 show that the test solutions and the control solutions are stable when left at room temperature (18-23 ℃) for 24 hours, and the method is highly practical.

2.3 Linear Range

Taking a proper amount of ethylenediamine, respectively adding ultrapure water to dilute into a series of solutions of 0.40 mu g/ml, 1.00 mu g/ml, 1.99 mu g/ml, 2.99 mu g/ml and 5.98 mu g/ml, respectively taking 25ul of the solutions to inject into an ion chromatograph, recording a chromatogram, and performing linear regression by taking the concentration of the ethylenediamine as a horizontal coordinate (x) and the area (y) of the chromatographic peak as a vertical coordinate, wherein the result is shown in figure 12, and the linear regression equation is as follows:

y＝0.0639x-0.0099(R²＝0.9976)

the results show that when the concentration of ethylenediamine is in the range of 0.4. mu.g/ml to 5.98. mu.g/ml, the concentration has a good linear relationship with the chromatographic peak area (r. 0.9988). The lowest linear detection concentration of the method for the ethylenediamine is 0.40 mu g/ml, and the detection linear range is 0.04 wt% -0.60 wt%.

2.4 detection Limit

Diluting the ethylenediamine reference substance solution with ultrapure water for multiple times, and then injecting samples respectively according to the signal-to-noise ratio of 3:1 calculation the minimum detected concentration of ethylenediamine was 0.06. mu.g/ml. The chromatogram at an ethylenediamine concentration of 0.06. mu.g/ml is shown in FIG. 13.

2.5 precision

The ethylene diamine control solution was measured 6 times in duplicate and the results are shown in Table 6 with an RSD of 1.48%. The precision of the method of the invention meets the requirement.

TABLE 6 results of precision test

Number of measurements	Retention time (min)	Peak area
			dz-1	10.18	0.092
dz-2	10.18	0.093
			dz-3	10.17	0.090
dz-4	10.18	0.094
			dz-5	10.16	0.092
dz-6	10.17	0.092
			Mean value of	10.17	0.090
RSD％	0.08％	1.48％

2.6 durability

Accurately weighing 99.73mg ethylenediamine, preparing stock solution with concentration of 9.97 μ g/ml with ultrapure water, and diluting the stock solution with ultrapure water to obtain 1.99 μ g/ml solution to obtain reference solution.

Precisely weighing 9.99mgXG-SM2, and preparing into solution with concentration of 999.00 mug/ml with ultrapure water to obtain the test solution.

According to the detection method of the ethylenediamine provided by the invention, the reference solution and the test solution are used, the same chromatographic column is used, the flow rate and the column temperature are respectively adjusted finely, and as a result, other components in the test sample have no interference on the detection of the ethylenediamine, which indicates that the built method has high durability. The results are shown in Table 7.

TABLE 7 durability test results

2.7 accuracy (sample recovery experiment)

98.24mg of ethylenediamine is precisely weighed, a stock solution with the concentration of 9.824 mug/ml is prepared by ultrapure water, then a proper amount of the stock solution is taken and diluted by ultrapure water to form a reference substance solution with the concentration of 1.96 mug/ml.

9 parts of sitagliptin phosphate intermediate XG-SM2, each of which is about 10mg, are precisely weighed and prepared into a test solution with the concentration of about 1mg/ml by using ultrapure water. Respectively and precisely absorbing 25 mu l of the mixture, injecting the mixture into an ion chromatograph, recording a chromatogram, and calculating the content of the ethylenediamine. Then, the stock solutions with the corresponding volumes were added (wherein 50% of the stock solutions were added to 1ml of the stock solutions, 100% of the stock solutions were added to 2ml of the stock solutions, and 300% of the stock solutions were added to 3ml of the stock solutions) as shown in table 8, after shaking up, 25 μ l of the stock solutions were precisely extracted, respectively injected into an ion chromatograph, measured, recorded in a chromatogram, calculated for the content of ethylenediamine, and calculated for the recovery rate, the results are shown in table 8, and the ion chromatogram of the sample solution to be tested for sample recovery with the number of 100% -1 is shown in fig. 14.

TABLE 8 sample recovery test results

Table 8 shows that the method of the invention has high accuracy, and FIG. 14 also shows that the sitagliptin phosphate intermediate XG-SM2 has no influence on the detection of ethylenediamine.

In conclusion, the methodology research proves that the method for detecting the free hydrazine in the sitagliptin phosphate by using the ion chromatography has the advantages of good specificity, stability and durability, high precision and accuracy, the lowest detected concentration determined by the signal-to-noise ratio of 3:1 is 0.06 mug/ml, and the lowest linear detected concentration is 0.4 mug/ml.

Example 3The method is used for measuring 2 batches of sitagliptin phosphate intermediate XG-SM2

The specific measurement results are as follows:

respectively weighing a proper amount of sitagliptin phosphate intermediate XG-SM2, adding ultrapure water for dissolving, and diluting to prepare a solution containing about 1mg of the sitagliptin phosphate intermediate XG-SM2 per 1ml, wherein the solution is used as a reference solution; an appropriate amount of ethylenediamine was precisely measured and diluted with ultrapure water to a solution containing about 2. mu.g per 1ml, which was used as a control solution. Under the chromatographic conditions described in example 2, 25. mu.l of the control solution was measured precisely and injected into an ion chromatograph, and the sensitivity of the apparatus was adjusted so that the peak height of the chromatogram of the principal component was 10% to 20% of the full scale. And precisely measuring 25 mu l of each of the test solution and the reference solution, respectively injecting the samples, injecting the samples into an ion chromatograph, recording the retention time of the chromatogram to the main component peak by 5 times, and calculating the content of ethylenediamine in the sitagliptin phosphate intermediate XG-SM2 according to an external standard method. The results are shown in Table 9.

Table 9 measurement results

Name (R)	Batch number	Sample weighing (mg)	Concentration (μ g/ml)	Peak area	Content of ethylenediamine
						Ethylene diamine		99.64	1.99	0.117	/
XG-SM2	C182016001	10.04	1004.00	0.046	0.08％
						XG-SM2	C182017013	10.02	1002.00	0.037	0.06％

In a word, the invention provides a method for detecting ethylenediamine based on ion chromatography, and further provides a method for qualitatively/quantitatively detecting ethylenediamine in sitagliptin phosphate key intermediate XG-SM2 based on ion chromatography. The method has strong specificity and high sensitivity, the lowest linear detection concentration is 0.4 mu g/ml, and the lowest detection concentration determined by the signal-to-noise ratio of 3:1 is 0.06 mu g/ml. Therefore, the detection method of the ethylenediamine provided by the invention can be used for effectively and quickly detecting the ethylenediamine in the sitagliptin phosphate intermediate XG-SM2, so that the quality of a product is better controlled, and the safety of clinical medication is guaranteed.

Claims (1)

1. A method for detecting ethylenediamine in a sitagliptin phosphate intermediate XG-SM2 with a structural formula shown as a formula (II),

comprises the steps of establishing chromatographic conditions, preparing a reference solution, preparing a test solution, qualitatively detecting and/or quantitatively determining, wherein,

establishment of chromatographic conditions:

a chromatographic column: thermo Dionex ion pac CS12A cation chromatography column, 250mm 4 mm;

and (3) protecting the column: thermo Dionex ion pac CG12 guard column, 4mm x 50 mm;

a detector: a conductance detector;

mobile phase: 40mmol/l methanesulfonic acid;

flow rate: 1.0 ml/min;

column temperature: at 32 ℃;

sample introduction amount: 25 mul;

preparation of control solutions:

precisely measuring a proper amount of ethylenediamine, dissolving with ultrapure water, and diluting into a solution containing 2 mu g of ethylenediamine per 1ml to obtain the ethylenediamine aqueous solution;

preparation of a test solution:

taking a proper amount of the sitagliptin phosphate intermediate XG-SM2, adding ultrapure water for dissolving, and diluting into a solution containing 1mg of the sitagliptin phosphate intermediate XG-SM2 per 1ml to obtain the sitagliptin phosphate intermediate XG-SM 2;

and (3) qualitative detection:

under the chromatographic condition, precisely measuring 25 mu l of reference solution, injecting the reference solution into an ion chromatograph, and adjusting the sensitivity of the detector to ensure that the chromatographic peak height of the main component is 10-20% of the full-scale range; precisely measuring 25 μ l of each of the test solution and the reference solution, injecting sample, injecting into ion chromatograph, and recording chromatogram; comparing the chromatogram with the chromatogram of the reference solution, and observing whether a corresponding chromatographic peak appears in the chromatogram of the test solution at a corresponding retention time;

and/or

Quantitative determination:

under the chromatographic condition, precisely measuring 25 mu l of reference solution, injecting the reference solution into an ion chromatograph, and adjusting the sensitivity of the detector to ensure that the chromatographic peak height of the main component is 10-20% of the full-scale range; and precisely measuring 25 mul of each of the test solution and the reference solution, injecting the samples respectively, injecting the samples into an ion chromatograph, and calculating the content of the ethylenediamine in the test solution according to an external standard method.

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