CN113484450A

CN113484450A - Derivatization treatment method for detecting enantiomer of drug, determination method and application

Info

Publication number: CN113484450A
Application number: CN202110807436.1A
Authority: CN
Inventors: 冯舟; 刘瑞霖; 赵永龙; 熊尧; 樊娇娇
Original assignee: Sichuan Pharmaceutical Inc
Current assignee: Sichuan Pharmaceutical Inc
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2021-10-08
Anticipated expiration: 2041-07-16
Also published as: CN113484450B

Abstract

The invention discloses a derivatization treatment method for detecting a drug enantiomer, a determination method and application. When the derivatization treatment method is used for detecting isomers in sitagliptin phosphate bulk drugs and preparations, the high performance liquid chromatography is adopted for detection, and the chromatographic conditions are as follows: performing gradient elution by using octadecylsilane chemically bonded silica as a filler, triethylamine aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate is 1.5ml per minute, and the detection wavelength is 340 nm; and (3) dissolving the sample by adopting a reverse phase system, and performing derivatization treatment by adopting a Marfey reagent. The method has the advantages that the sitagliptin phosphate bulk drug and the enantiomer have higher separation degree, the problems of longer preparation time, poorer reproducibility and high cost of the test product in the prior detection technology can be solved, the specificity is strong, the accuracy is strong, the precision is high, the durability is good, the operation is convenient, and the quality of the bulk drug and the preparation can be effectively controlled in the actual production.

Description

Derivatization treatment method for detecting enantiomer of drug, determination method and application

Technical Field

The invention relates to the technical field of drug analysis, in particular to a derivatization treatment method for detecting a drug enantiomer, a determination method and application.

Background

EP10.0 and USP40 disclose a method for detecting enantiomers in sitagliptin phosphate bulk drugs and preparations, but there is still a problem in practical detection, and although the separation degree of enantiomers and main peaks is good, a chromatographic column for chiral separation is a normal phase type chromatographic column in which the surface of silica gel is coated with amylose (chromatographic column 250mm × 4.6mm, 5 μm), and in the chromatographic condition, 0.1% of water is added to a mobile phase, and 10% of water is also added to a diluent used in the preparation of a sample. It is well known that a normal phase chiral column cannot use water as a mobile phase, nor can it dissolve a sample with water. Otherwise the column will be damaged. In the actual detection of the sitagliptin phosphate bulk drug and the preparation, particularly when a preparation sample is detected, the column efficiency of a chiral chromatographic column is extremely easy to reduce, the column pressure is extremely easy to increase, the chromatographic column cannot be used, and the detection of enantiomers can be influenced. Resulting in increased detection costs.

The patent with publication number CN 109580833A discloses a method for measuring enantiomers in a sitagliptin phosphate raw material and a preparation, and discloses a method which adopts chiral crown ether silica gel as a filling agent and adopts a perchloric acid solution and acetonitrile mixed solution as a mobile phase; the sample is prepared from a water solvent sitagliptin phosphate raw material and a preparation. The method adopts chiral crown ether silica gel as a filling agent, and avoids the damage of water for sample preparation to a chromatographic column. The method is rapid, the whole separation process is 15min, and the separation degree of the two components is greater than 1.5, which meets the requirement of related chromatography on the separation degree. However, the method is not good, and in the method, the sitagliptin phosphate raw material is taken and dissolved in a diluent and diluted to be a solution with the concentration of 1mg/mL, and then the solution is directly placed at minus 80 ℃ for 2 hours and then placed at 25-28 ℃ for 1 hour to obtain a test solution. The preparation time of the test sample is long, the reproducibility is poor, the chromatographic column using the chiral crown ether silica gel as the filler is expensive, and the detection of the finished product is still high.

Disclosure of Invention

The invention aims to provide a derivatization treatment method for detecting a drug enantiomer, which is used for detecting an S-enantiomer in a sitagliptin phosphate bulk drug and a preparation, so that the bulk drug and the enantiomer have higher separation degree, and the problems of longer preparation time, poorer reproducibility and high cost of a test product in the prior detection technology can be solved.

The invention is realized by the following technical scheme:

a derivatization treatment method for detecting the enantiomer of a medicine comprises the steps of adding a Marfey reagent solution into a solution to be derivatized, adding a sodium bicarbonate solution, sealing a reaction bottle, carrying out mixing and derivatization on the reaction bottle in a constant-temperature water bath at 35-55 ℃ for 0.5-1.5 hours, adding a hydrochloric acid solution, and fully mixing to stop reaction.

The derivatization processing method for detecting the drug enantiomer is used for detecting the enantiomer in the sitagliptin phosphate bulk drug and the preparation.

The determination method of the enantiomers in the sitagliptin phosphate bulk drug and the preparation adopts the high performance liquid chromatography for detection, and the chromatographic conditions are as follows:

performing gradient elution by using octadecylsilane chemically bonded silica as a filler, triethylamine aqueous solution as a mobile phase A and acetonitrile as a mobile phase B at a flow rate of 1.5ml +/-0.1 ml per minute and a detection wavelength of 340nm +/-2 nm;

and (3) dissolving the sample by adopting a reverse phase system, and performing derivatization treatment by adopting a Marfey reagent.

The Marfey reagent is a chiral reagent, and is [ N alpha- (2, 4-dinitro-5-fluorophenyl) -L-alaninamide ].

According to the invention, a reverse phase system is adopted for dissolving the sitagliptin phosphate raw material and preparation, and the chiral reagent Marfey reagent [ N alpha- (2, 4-dinitro-5-fluorophenyl) -L-alaninamide ] is introduced for pre-column derivatization of the sample, so that the sample preparation time can be effectively shortened, the reproducibility is good, and meanwhile, the sample solution has high stability.

The invention adopts octadecylsilane chemically bonded silica as a filling agent, has the advantage of low price and reduces the cost compared with chiral crown ether silica.

In conclusion, compared with the prior art, the method has the advantages that the separation degree of the raw material medicine and the impurities is higher, and the problems of longer preparation time, poorer reproducibility and high cost of the test product in the prior detection technology can be solved.

The invention has strong specificity, strong accuracy, high precision, good durability and convenient operation, and can effectively control the quality of the bulk drugs and the preparations in the actual production.

Further, the mobile phase A is 0.5-1.0% of triethylamine aqueous solution, and the pH value is adjusted to 1.0-3.0 by adopting an acidic solution.

Further, the acidic solution is phosphoric acid.

Further, the specific procedure of gradient elution is shown in the following table:

further, a diluent is adopted to dissolve samples, wherein the samples comprise a test sample, a sitagliptin phosphate reference substance and a sitagliptin S-enantiomer reference substance.

Further, the diluent is a solution of water and acetonitrile mixed in a volume ratio of 1: 1.

Further, the specific process of the derivatization treatment is as follows:

transferring the solution to be derivatized into a reaction bottle, adding a Marfey reagent solution, adding a sodium bicarbonate solution, sealing the reaction bottle, carrying out mixing and derivatization on the reaction bottle in a constant-temperature water bath at 35-55 ℃ for 0.5-1.5 hours, adding a hydrochloric acid solution, and fully mixing to stop the reaction.

Sodium bicarbonate solution provided the reaction conditions and hydrochloric acid solution was added to stop the reaction.

Further, the molar ratio of the Marfey reagent solution to the solution to be derivatized is 1-2, and the concentration of the Marfey reagent solution is 5 g/L.

Further, the molar ratio of the sodium bicarbonate solution to the hydrochloric acid solution is 1:1, and the concentrations of the hydrochloric acid solution and the sodium bicarbonate solution are both 1 mol/L.

The effect of the addition of the Marfey reagent on the component content is shown in table 1:

TABLE 1

Addition amount of Marfey reagent	Content of Main component (%)	Isomer content (%)
			0.8ml	99.728	0.272
1.0ml	99.729	0.271
			1.2ml	99.728	0.272
1.5ml	99.728	0.272

As can be seen from Table 1, since 0.8ml of the Marfey reagent is added in a molar ratio of 1:1, when the amount is excessive, there is substantially no effect on the measurement results, but too little of the Marfey reagent may affect the accuracy of the content of the isomer in the measurement results.

Further, the method comprises the following steps:

s1, sample preparation:

test solution: dissolving and diluting a sample with a diluent to prepare a solution containing 6mg of sitagliptin in each 1ml, shaking up, centrifuging a proper amount, and taking supernatant;

system applicability solution: respectively and precisely weighing a sitagliptin phosphate reference substance and a sitagliptin isomer reference substance, dissolving and diluting by using a diluent to prepare a mixed solution containing 6mg of sitagliptin and 9 mu g of isomer in each 1ml,

the diluent is a solution mixed by water and acetonitrile according to the volume ratio of 1: 1;

s2, derivatization treatment:

transferring a solution to be derivatized into a reaction bottle, adding a Marfey reagent solution, adding a sodium bicarbonate solution, sealing the reaction bottle, carrying out mixing and derivatization on the reaction bottle in a constant-temperature water bath at 35-55 ℃ for 0.5-1.5 hours, adding a hydrochloric acid solution, and fully mixing to stop reaction;

the solution to be derivatized comprises a test solution, a system applicability solution and a blank solution;

the effect of temperature on the content of ingredients in the derivatization treatment is shown in table 2:

TABLE 2

Temperature of Water bath (. degree.C.)	Content of Main component (%)	S-enantiomer content (%)
			35	99.734	0.266
40	99.729	0.271
			45	99.727	0.273
50	99.721	0.279
			55	99.705	0.295

As can be seen from Table 2:

constant temperature water bath is carried out at 35-55 ℃, and when the temperature of the water bath is 35-55 ℃, the detection result is basically not influenced.

The effect of time on the content of components in the derivatization treatment is shown in table 3:

TABLE 3

Time of water bath (hours)	Content of Main component (%)	S-enantiomer content (%)
			0.5	99.730	0.270
0.8	99.731	0.269
			1.0	99.729	0.271
1.2	99.727	0.273
			1.5	99.727	0.273

As can be seen from Table 3:

when the water bath time is 0.5 to 1.5 hours, the detection result is basically not influenced.

S3, detection:

setting chromatographic conditions, precisely sucking 5-100 mul of system applicability solution and sample solution respectively, injecting samples respectively, and recording chromatograms.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the method can not only accurately determine the absolute configuration of sitagliptin phosphate and the S-enantiomer thereof, but also has high sensitivity.

2. By adopting derivatization treatment, the test solution has higher stability, and experiments prove that the stability is good within 90 hours.

3. The invention can separate the sitagliptin or the derivative of the sitagliptin salt from the derivative of S-enantiomer of the sitagliptin, and effectively control the quality of the sitagliptin phosphate bulk drug and the preparation thereof; the method is simple and convenient to operate, easy to control, low in detection cost, good in specificity and precision, and accurate and reliable in detection result, provides an effective detection method for monitoring the content of the S-enantiomer in the sitagliptin phosphate medicine, and further ensures the quality of the sitagliptin phosphate medicine and the medication safety of patients.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a detection chromatogram of example 1;

FIG. 2 is a linear plot of the isomers of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example 1:

the determination method of the enantiomers in the sitagliptin phosphate bulk drug and the preparation adopts the high performance liquid chromatography for detection,

instruments and reagents: a liquid chromatograph, an electronic balance, a pH meter, a water bath, triethylamine, phosphoric acid, acetonitrile, a Marfey reagent, sodium bicarbonate and hydrochloric acid;

chromatographic conditions are as follows:

octadecylsilane chemically bonded silica was used as a filler, a 0.72% triethylamine aqueous solution (pH adjusted to 2.0 with phosphoric acid) was used as a mobile phase a, and acetonitrile was used as a mobile phase B, and gradient elution was performed according to table 1 using water: acetonitrile (50:50) as a diluent, at a flow rate of 1.5ml per minute, the detection wavelength was 340 nm.

TABLE 1

The specific operation process comprises the following steps:

s1, sample preparation:

test solution: a test sample was taken, and diluted with a diluent [ water: dissolving acetonitrile (50:50) ] and diluting to obtain a solution containing about 6mg of sitagliptin in each 1ml, shaking up, centrifuging a proper amount, and taking supernatant to obtain the sitagliptin-N-methyl-ethyl-L-arginine salt;

system applicability solution: respectively taking a proper amount of a sitagliptin phosphate reference substance and a proper amount of a sitagliptin isomer reference substance, precisely weighing, and adding a diluent [ water: acetonitrile (50:50) ] is dissolved and diluted to prepare a mixed solution containing about 6mg of sitagliptin and about 9 mu g of isomer in each 1ml, and the preparation is obtained;

s2, derivatization treatment:

2ml of the solution to be derivatized are transferred to a 10ml reaction vial, 1.0ml of 5g/L Marfey reagent solution in acetonitrile are added, 100. mu.l of sodium bicarbonate solution (1mol/L) are added, the vial is sealed, the vial is subjected to a thermostatic water bath at 45 ℃ for 1.0 hour, mixing and derivatization are carried out. About 100. mu.l of hydrochloric acid solution (1mol/L) was further added thereto, and mixed well to stop the reaction. Adding the mobile phase to a constant volume to scale, and shaking up;

the solution to be derivatized comprises a test solution, a system suitability solution and a blank solution (diluent);

the principle is as follows:

s3, detection:

setting chromatographic conditions, precisely sucking 20 μ l of each of the system applicability solution and the sample solution, injecting sample, and recording chromatogram as shown in FIG. 1.

In fig. 1, RT ═ 36.378 sitagliptin derivatives; RT-23.129 sitagliptin isomer derivatives.

The parameters of the peak area and the degree of separation of FIG. 1 are shown in Table 4:

TABLE 4

1) The linear range is good:

specific test methods for linear range:

linear solutions (both dissolved with diluent to constant volume): precisely weighing 2.5mg → 25ml of S-enantiomer reference substance to obtain S-enantiomer stock solution (with concentration of about 100 μ g/ml);

linear 1: weighing 79mg of sitagliptin phosphate working reference substance into a 10ml measuring flask precisely, adding 6ml of S-enantiomer stock solution (the concentration of S-enantiomer is about 60 mu g/ml and is 200%)

Linearity 2: a79 mg to 10ml measuring flask of a sitagliptin phosphate working control is precisely weighed, 4.5ml of S-enantiomer stock solution (the concentration of the S-enantiomer is about 45 mu g/ml and is 150 percent) is added

Linearity 3: weighing 79mg of sitagliptin phosphate working reference substance into a 10ml measuring flask precisely, adding 3ml of S-enantiomer stock solution (the concentration of the S-enantiomer is about 30 mu g/ml and is 100 percent)

Linearity 4: a79 mg to 10ml measuring flask of a sitagliptin phosphate working control is precisely weighed, and 1.5ml of S-enantiomer stock solution (the concentration of the S-enantiomer is about 15 mu g/ml and is 50 percent) is added

Linearity 5: weighing 79mg of sitagliptin phosphate working reference substance into a 10ml measuring flask precisely, adding 0.9ml of S-enantiomer stock solution (the concentration of S-enantiomer is about 9 mu g/ml, 30%)

Linearity 6: weighing 79mg of sitagliptin phosphate working control accurately into a 10ml measuring flask, adding 0.6ml of S-enantiomer stock solution (the concentration of S-enantiomer is about 6 mu g/ml, 20%)

Linearity 7: accurately weighing 79mg of sitagliptin phosphate working reference substance into a 10ml measuring flask, adding 0.3ml of S-enantiomer stock solution (the concentration of the S-enantiomer is about 3 mu g/ml and is 10 percent)

Linearity 8: quantitative limiting solution

A derivatization step: 2ml of the solution to be derivatized are transferred into a 10ml reaction vial, 2ml of 5g/L Marfey reagent solution in acetonitrile are added, 200. mu.l sodium bicarbonate solution (1mol/L) are added, the vial is sealed, and the vial is incubated in a thermostatic water bath at 45 ℃ for 1 hour, mixed and derivatized. About 200. mu.l of hydrochloric acid solution (1mol/L) was further added thereto, and mixed well to stop the reaction. Adding the mobile phase to a constant volume to scale, and shaking up.

③ derivatization: and (3) derivatizing the blank solvent and each linear solution according to a derivatization step to obtain each derivatization solution.

Fourthly, sample injection solution: blank derivatization solution has at least 1 needle, and each linear derivatization solution has 1 needle.

Simulating a linear regression curve by using the peak area ordinate and the concentration abscissa;

sixthly, the result requires that: the correlation coefficient (r) of the regression line should not be less than 0.999, and the Y-intercept should be within 25% of the 100% response value.

The results of the linearity and range of impurity F (S-enantiomer) are shown in table 5 and fig. 2:

TABLE 5

As can be seen from Table 5:

the S-enantiomer has a good linear relation between the concentration of 0.72 mu g/ml and 57.72 mu g/ml, the correlation coefficient of a linear regression equation is more than 0.999, the response value of the Y-axis intercept 100% is less than 2%, and the requirement is met.

2) Quantitative limit and detection limit:

the limit of quantitation of the S-enantiomer was 14.43ng (equivalent to 0.012% of the test article), and the limit of detection was 4.82ng (equivalent to 0.004% of the test article); the quantitative limit solution continuous sampling 6 needles have the peak area RSD of 5.41 percent and the retention time RSD of 0.20 percent, and the detection limit solution continuous sampling 6 needles have the peak area RSD of 5.05 percent and the retention time RSD of 0.21 percent, which all meet the verification requirements.

And (3) diluting the low-concentration sample under the linear term step by step to prepare a detection limit solution and a quantitative limit solution, injecting the sample according to a formulated method, and taking the concentration when the signal-to-noise ratio S/N is about 10, namely the quantitative limit concentration. And taking the concentration when the signal-to-noise ratio S/N is about 3, namely the detection limit concentration. Taking a quantitative limiting solution, and continuously injecting samples for 6 times, wherein the RSD of the retention time is less than 1 percent, and the RSD of the peak area is less than 10 percent; and taking the detection limiting solution, and continuously injecting for 6 times, wherein the RSD of the retention time is less than 1%, and the RSD of the peak area is less than 20%.

The results of the quantitation limit and detection limit are shown in table 6:

TABLE 6

The results of the quantitation limit solution precision are shown in table 7:

TABLE 7

The results of the precision of the detection limiting solution are shown in Table 8

TABLE 8

And (4) conclusion: the retention time RSD of the solution precision of the quantification limit and the detection limit is less than 1 percent, and the peak area RSD is less than 10 percent, thereby meeting the requirements.

3) And stability:

the testing process comprises the following steps: and respectively detecting the stability of the sample solution and the derivative solution of the standard solution.

31) Sample solution-100 mg: taking a test sample (batch number: 210303) (specification 100mg), grinding into powder, taking about 240mg → 10ml of the test sample which is ground into powder, dissolving with diluent by ultrasonic and diluting to scale, thus obtaining the sitagliptin (about 6mg in each 1 ml).

32) And the sample solution is 50 mg: taking a test sample (batch number: 210301) (specification 50mg), grinding into powder, taking about 240mg → 10ml of the test sample which is ground into powder, dissolving with diluent by ultrasonic and diluting to scale, thus obtaining the sitagliptin (1 ml contains about 6mg of sitagliptin).

33) And adding standard solution to the sample to be tested to 100 mg: taking a test sample (batch number: 210303) (specification 100mg), grinding into powder, taking about 240mg → 10ml of the test sample which is ground into powder, adding 3ml of the S-enantiomer stock solution, ultrasonically dissolving by using a diluent and diluting to scale, thus obtaining the sitagliptin (1 ml contains about 6mg of sitagliptin and about 30 mu g of S-enantiomer).

34) And adding standard solution to the sample to be tested to 50 mg: taking a test sample (batch number: 210301) (specification 50mg), grinding into powder, taking about 240mg → 10ml of the test sample which is ground into powder, adding 3ml of the S-enantiomer stock solution, ultrasonically dissolving by using a diluent and diluting to scale, thus obtaining the sitagliptin (1 ml contains about 6mg of sitagliptin and about 30 mu g of S-enantiomer).

35) And a derivatization step: 1ml of the solution to be derivatized is transferred into a 5ml reaction vial, 1ml of 5g/L Marfey reagent solution in acetonitrile is added, 100. mu.l of sodium bicarbonate solution (1mol/L) are added, the vial is sealed, and the vial is incubated in a thermostated water bath at 45 ℃ for 1 hour, mixed and derivatized. About 100. mu.l of hydrochloric acid solution (1mol/L) was further added thereto, and mixed well to stop the reaction. Adding the mobile phase (A: B: 50) to the constant volume to scale, and shaking up.

36) And derivatization: and performing derivatization on the blank solvent, the system applicability solution, the test article solution and the test article added standard solution according to a derivatization step to obtain each derivatization solution.

37) And sample injection solution: blank derivatization at least solution 1 needle, system applicability derivatization solution 1 needle, and adding standard derivatization solution 1 needle for each sample at each time point.

And (4) counting results: according to the same method for investigating the stability of the system applicability solution, the RSD of each peak area in the sample standard-added derivative solution is not more than 2 percent,

the test results of the solution stability test of the test articles are shown in Table 9 (25 ℃):

TABLE 9

And (4) conclusion: the RSD of the peak area% of the S-enantiomer derivative of the test solution with the specification of 100mg and the specification of 50mg is not more than 2% within 90 hours at 25 ℃, namely the sitagliptin phosphate enantiomer test solution is stable within 90 hours at 25 ℃.

The test results of the stability test of the test sample solutions (25 ℃) are shown in Table 10:

watch 10

And (4) conclusion: the RSD of the S-enantiomer derivative peak area% of the standard solution for the test article with 100mg specification and 50mg specification is not more than 2% at 25 ℃ within 90 hours, namely the S-enantiomer test article standard solution for the sitagliptin phosphate tablets is stable at 25 ℃ within 90 hours.

Example 2:

the influence of the small change of the chromatographic condition on the detection result is investigated

Adding a standard solution to a sample to be tested, wherein the standard solution is-100 mg: taking a test sample (batch number: 210303) (specification 100mg), grinding into powder, taking about 240mg → 10ml of the test sample which is ground into powder, adding 3ml of the S-enantiomer stock solution, dissolving with diluent by ultrasound, and diluting to scale to obtain the product (each 1ml contains about 6mg of sitagliptin and about 30 μ g of S-enantiomer).

Adding standard solution to the sample to be tested, wherein the standard solution is-50 mg: taking a test sample (batch number: 210301) (specification 50mg), grinding into powder, taking about 240mg → 10ml of the test sample which is ground into powder, adding 3ml of the S-enantiomer stock solution, ultrasonically dissolving by using a diluent and diluting to scale, thus obtaining the sitagliptin (1 ml contains about 6mg of sitagliptin and about 30 mu g of S-enantiomer).

③ blank auxiliary material solution-100 mg: taking about 163mg of blank adjuvant (batch number: 210512-1) (specification 100mg), precisely weighing, placing in 10ml measuring flask, dissolving with diluent and diluting to scale, shaking, centrifuging at 6000 rpm for 10 min, and collecting supernatant.

Fourthly, blank auxiliary material solution-50 mg: taking about 163mg of blank adjuvant (batch number: 210512-2) (specification 50mg), precisely weighing, placing in 10ml measuring flask, dissolving with diluent and diluting to scale, shaking, centrifuging at 6000 rpm for 10 min, and collecting supernatant.

Derivatization step: 1ml of the solution to be derivatized is transferred into a 5ml reaction vial, 1ml of 5g/L Marfey reagent solution in acetonitrile is added, 100. mu.l of sodium bicarbonate solution (1mol/L) are added, the vial is sealed, and the vial is incubated in a thermostated water bath at 45 ℃ for 1 hour, mixed and derivatized. About 100. mu.l of hydrochloric acid solution (1mol/L) was further added thereto, and mixed well to stop the reaction. Adding the mobile phase (A: B: 50) to the constant volume to scale, and shaking up.

Sixthly, derivatization: and performing derivatization on a blank solvent, a system applicability solution, a sample adding standard solution and a blank auxiliary material solution according to a derivatization step to obtain each derivatization solution.

Seventhly, sample injection solution: blank derivatization under each durability condition at least 1 needle of solution, system applicability derivatization under each durability condition at least 1 needle of solution, each test sample under each durability condition with 1 needle of standard derivatization solution, each blank adjuvant derivatization solution under each durability condition at least 1 needle.

And counting results: the blank solvent and the blank auxiliary materials do not interfere with the detection of the sample; the isomer content RSD under each durability condition is not more than 2%

The durability test results are shown in table 11:

TABLE 11

And (4) conclusion: since the isomer was not detected in the product, a durability test was conducted using a standard sample. The content RSD of the isomers of the sample added standard solution is less than 2 percent under various durability conditions of samples with different specifications, and the requirements are met.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The derivatization treatment method for detecting the drug enantiomer is characterized in that a Marfey reagent solution is added into a solution to be derivatized, then a sodium bicarbonate solution is added, a reaction bottle is sealed, the reaction bottle is put in a constant-temperature water bath at 35-55 ℃ for 0.5-1.5 hours for mixing and derivatization, then a hydrochloric acid solution is added, and the mixture is fully mixed to stop the reaction.

2. The derivatization treatment method for detecting the drug enantiomer of claim 1 is used for detecting the S-enantiomer in a sitagliptin phosphate bulk drug and preparation.

3. The method for measuring the S-enantiomer in the sitagliptin phosphate bulk drug and the preparation adopts high performance liquid chromatography for detection, and is characterized in that the chromatographic conditions are as follows:

the sample is prepared by dissolution using a reverse phase system and derivatization according to claim 1 using Marfey's reagent.

4. The method for determining S-enantiomer in sitagliptin bulk drugs and preparations according to claim 3, characterized in that the mobile phase A is 0.5% -1.0% triethylamine aqueous solution, and the pH value is adjusted to 1.0-3.0 by adopting acidic solution.

5. The method for determining S-enantiomer in sitagliptin bulk drug and preparation according to claim 4, characterized in that the acidic solution is phosphoric acid.

6. The method for determining S-enantiomer in sitagliptin bulk drug and preparation according to claim 3, characterized in that the specific process of gradient elution is as shown in the following table:

7. the method for determining S-enantiomer in sitagliptin bulk drugs and preparations according to claim 3, characterized in that the molar ratio of the Marfey reagent solution to the solution to be derivatized is 1-2, and the concentration of the Marfey reagent solution is 5 g/L.

8. The method for determining S-enantiomer in sitagliptin bulk drug and preparation according to claim 3, wherein the molar ratio of the sodium bicarbonate solution to the hydrochloric acid solution is 1:1, and the concentrations of the hydrochloric acid solution and the sodium bicarbonate solution are both 1 mol/L.

9. The method for determining S-enantiomer in sitagliptin bulk drug substances and preparations according to any one of claims 1 to 8, characterized by comprising the following steps:

s1, sample preparation:

system applicability solution: respectively and precisely weighing a sitagliptin phosphate reference substance and a sitagliptin S-enantiomer reference substance, dissolving and diluting by using a diluent to prepare a mixed solution containing 6mg of sitagliptin and 9 mu g of S-enantiomer in each 1ml,

s2, derivatization treatment:

s3, detection: