CN103175927A

CN103175927A - Method for content determination and impurity determination of taltirelin and preparation thereof

Info

Publication number: CN103175927A
Application number: CN2011104333679A
Authority: CN
Inventors: 李志刚; 顾群; 孙学伟; 宋华付
Original assignee: BENCAO TIANYUAN PHARMACEUTICAL RESEARCH INST BEIJING
Current assignee: BENCAO TIANYUAN PHARMACEUTICAL RESEARCH INST BEIJING
Priority date: 2011-12-21
Filing date: 2011-12-21
Publication date: 2013-06-26

Abstract

The invention relates to an analysis method of a medicament, and particularly relates to a method for content determination and impurity determination of taltirelin by high-performance liquid chromatography. A high-performance liquid chromatography detection method, which is good in separation effect, high in sensitivity, and fast analyzes the contents and impurities of the taltirelin and the preparation thereof, is built under the detection conditions that octadecylsilane chemically bonded silica is taken as a filler, and acetonitrile-(0.001mol/L to 0.10mol/L) phosphate buffered solution ((30 to 70) to (10 to 90)) is used as a mobile phase. By adopting the method, the change of the impurities in the processes of producing and storing the taltirelin can be effectively detected; and the method has important practical significance for quality control of the medicament.

Description

The assay of a kind of Taltirelin and preparation thereof and the method for impurity determination

Technical field

The present invention relates to a kind of analytical approach of medicine, particularly relate to a kind of with high-performance liquid chromatogram determination Taltirelin and the assay of preparation and the method for impurity determination.

Background technology

Taltirelin, chemical name: (-)-N-[(S)-six hydrogen-1-methyl-2,6-dioxo-4-pyrimidine radicals carbonyl]-L-histamine acyl-L-prolineamide tetrahydrate, it is the oral thyrotrophin-releasing hormone (TRH) of first approval in the world, except having the endocrine effect, also can bring into play certain central nervous system (CNS) effect, comprise the raising locomotor activity, body temperature that the antagonism Reserpine is induced reduces, and the sleep of inducing of antagonism amobarbital.This medicine is by the exploitation of day Honda limit drugmaker, and go on the market in Japan in July, 2000, is used for improving patient's SCD incoordination.Pharmaceutical research shows that Taltirelin produces strong and lasting multiple action via brain TRH acceptor to CNS.This medicine is stronger 10～100 times than TRH to the excitation of CNS, and acting duration is about 8 times than TRH; Affinity to the TRH acceptor is about 1/11 of TRH, thus its endocrine effect than TRH a little less than, but Taltirelin is more stable than TRH in vivo.In addition, the 1/6-1/11 that act as TRH of Taltirelin to thyroid-stimulating hormone (TSH) release; Central nervous system is had stronger effect, but its hormone-like effect is less simultaneously, so spinoff is less.Clinical studies show, the curative effect of 5mg Taltirelin is better, and better tolerance, and bad reaction is slight.Taltirelin will be brought into play huger effect in the clinical practice of neurodegenerative disease.In view of the advantage of Taltirelin at the treatment neurodegenerative disease, and there is no at home this kind of manufacturer production.China's independent research and produce this medicine for safeguarding China's people's health, and increases social benefit, economic benefit aspect and all is significant.

The Taltirelin molecular structural formula is:

Due to a kind of medicine from the synthesis material medicine to the relevant preparation of preparation, again through storage, transportation, use, experience one section comparatively complicated and very long process, each process all may produce relevant impurity during this period, may bring initiation material, reagent, intermediate, accessory substance and isomeride etc. in producing; May produce the special impurities such as catabolite, polymkeric substance or crystal transfer in storage and transportation.The process contaminants that impurity in medicine general reference produces in the production of medicine and storage and transport process or catabolite etc.The bad reaction that medicine produces in clinical use also has much relations except outside the Pass the pharmacologically active with major component has with the impurity that exists in medicine, and in medicine, the control of impurity is an importance of drug research and development, is also the guarantee of clinical safety in utilization.Therefore, in order to guarantee the safe and effective of medicine, also to consider the production actual conditions simultaneously, all impurity be detected as the important indicator of controlling drug quality in the research process of medicine both at home and abroad.It is one of thin spot in present China drug research and development that medicine impurity detects research.Want General Promotion China drug research and development level, conscientiously guarantee the security of public's medication, must pay attention to and strengthen the research of relative substance in medicine.

Find after deliberation, Taltirelin and preparation thereof issuable impurity in the process of preparation and accumulating thereof has:

Impurity 1 impurity 2 impurity 3

By literature search, yet there are no the report of the detection method of the detection method of content of Taltirelin and preparation thereof and impurity.Affect to a certain extent production and the application of Taltirelin and preparation thereof, demand studying the content of Taltirelin and preparation thereof and the determination method of impurity urgently.

Summary of the invention

The purpose of this invention is to provide a kind of good separating effect, highly sensitive, analyze quick Taltirelin and the content of preparation and the high-efficiency liquid chromatography method for detecting of impurity.

To achieve the above object of the invention, the present invention realizes by the following technical solutions:

The assay of a kind of Taltirelin and preparation thereof and the efficient liquid-phase chromatography method of impurity determination, it is characterized in that the chromatographic column take octadecylsilane chemically bonded silica as filling agent, so that acetonitrile-(phosphate buffered solution (30: 70～10: 90) of 0.001mol/L～0.10mol/L) is as mobile phase.

Wherein,

Preferred 15: 85 of the ratio of described acetonitrile-phosphate buffered solution.

The preferred 0.01mol/L of the concentration of described phosphate buffered solution.

Described phosphate is selected from one or more in sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate or dipotassium hydrogen phosphate.

Preferably contain the ion-pairing agent that concentration is 1～10g/1000mL in described phosphate buffered solution.

Described ion-pairing agent is selected from one or more in sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, perfluorooctane sulfonate, decane sodium sulfonate.

The pH value of the described phosphate buffered solution that contains ion-pairing agent is preferred 1～5, and more preferably 2.5.

The below simply introduces research process of the present invention:

(1) the chromatogram testing conditions determines

1. detect the initial option of wavelength

Get Taltirelin, impurity 1, impurity 2, impurity 3 reference substances appropriate, add the methyl alcohol dissolving, measure according to UV-VIS spectrophotometry (two appendix IV A of Chinese Pharmacopoeia version in 2005), the results are shown in Table 1:

The uv absorption of table 1 Taltirelin and impurity thereof

Title	Uv absorption
		Taltirelin	204nm, end absorbs
Impurity 1	203nm, end absorbs
		Impurity 2	201nm, end absorbs
Impurity 3	207nm, end absorbs

Conclusion: as shown in Table 1, the absorption region of Taltirelin, impurity 1, impurity 2, impurity 3 is at 200nm～210nm, therefore the detection wavelength of Taltirelin content and impurity determination is decided to be 205nm ± 5nm.

2. the selection and optimization of mobile phase

At first, the inventor regulates the ratio of acetonitrile and water take acetonitrile-water as basic mobile phase, and the mobile phase of Taltirelin content and impurity determination is selected in research.Result of study sees Table 2:

The initial option of table 2 mobile phase

Mobile phase composition/proportioning	System evaluation
		Acetonitrile-water (10: 90)	Taltirelin and impurity overlap with solvent peak
Acetonitrile-water (20: 80)	Taltirelin and impurity are difficult to separate with solvent peak
		Acetonitrile-water (30: 70)	Taltirelin and impurity are difficult to separate (accompanying drawing Fig. 1) with solvent peak
Acetonitrile-water (40: 60)	Taltirelin and impurity retention time are too short, and main peak is difficult to separate with impurity peaks

Conclusion: by upper table result as seen, acetonitrile-water (40: 60) is all very short as the retention time of mobile phase main peak and impurity, and main peak is difficult to separate with impurity peaks.Suitable as the mobile phase retention time with acetonitrile-water (30: 70～10: 90), but be difficult to separate with solvent peak.

The inventor continues flow and is optimized mutually, take acetonitrile-phosphate buffered solution as basic mobile phase, regulates the ratio of acetonitrile and phosphate buffered solution, optimizes mobile phase with this.Phosphate wherein is selected from one or more in sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate or dipotassium hydrogen phosphate.Result of study sees Table 3:

The initial optimization of table 3 mobile phase

Mobile phase composition/proportioning	System evaluation
		Acetonitrile-0.001mol/L phosphate buffered solution (10: 90)	Main peak, impurity peaks, solvent peak do not overlap, but the peak type is poor
Acetonitrile-0.10mol/L phosphate buffered solution (10: 90)	The same
		Acetonitrile-0.001mol/L phosphate buffered solution (30: 70)	The same
Acetonitrile-0.10mol/L phosphate buffered solution (30: 70)	The same
		Acetonitrile-0.01mol/L phosphate buffered solution (20: 80)	The same
Acetonitrile-0.01mol/L phosphate buffered solution (15: 85)	The same

Conclusion: by upper table result as seen so that acetonitrile-(phosphate buffered solution (30: 70～10: 90) of 0.001mol/L～0.10mol/L) is as mobile phase, the peak-to-peak degree of separation of Taltirelin and impurity and solvent makes moderate progress but the peak type is relatively poor, still needs further to optimize.

The inventor continues flow and is optimized mutually, adds ion-pairing agent in phosphate buffered solution, and the pH value of adjusting buffer solution is improved the peak type of Taltirelin and impurity thereof with this.Concrete steps are: take a certain amount of ion-pairing agent, be dissolved in the 1000mL phosphate buffered solution.Described ion-pairing agent wherein is selected from one or more in sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, perfluorooctane sulfonate, decane sodium sulfonate.Result of study sees Table 4:

Table 4 is further optimized mobile phase with ion-pairing agent

Conclusion: by upper table data as can be known, take pH to 1～5, ion-pairing agent concentration is as the phosphate solution of 1～10g/1000mL, phosphate concn 0.001mol/L～0.10mol/L as buffer solution, take acetonitrile as organic phase, wherein buffer solution and acetonitrile ratio in the mobile phase system of 30: 70～10: 90 scopes, can realize the detection analysis of Taltirelin and impurity thereof preferably.

3. auxiliary material interference experiment

The inventor also adopts technical solution of the present invention that the pharmaceutic adjuvant of preparation Taltirelin preparation commonly used on market is studied, and finds that after deliberation pharmaceutic adjuvant commonly used does not disturb the technical program.

(2) special impurities detection side science of law research

1. to the Taltirelin methodological study

(1) chromatographic condition: be filling agent with octadecylsilane chemically bonded silica, the detection wavelength is 205nm, with the sodium dihydrogen phosphate buffer (10: 90) of acetonitrile-(0.001mol/L) as mobile phase.

By above-mentioned chromatographic condition, the Taltirelin reference substance solution is measured, with the peak area of each Taltirelin, sample size is carried out linear regression.Under this chromatographic system, Taltirelin is good in 3.28～7.61 μ g/ml concentration range internal linear relations, and the detection of Taltirelin is limited to 6.56ng.

(2) chromatographic condition: be filling agent with octadecylsilane chemically bonded silica, the detection wavelength is 210nm, with the dipotassium hydrogen phosphate buffer solution (30: 70) of acetonitrile-(0.10mol/L) as mobile phase.

By above-mentioned chromatographic condition, the Taltirelin reference substance solution is measured, with the peak area of each Taltirelin, sample size is carried out linear regression.Under this chromatographic system, Taltirelin is good in 1.21～5.89 μ g/ml concentration range internal linear relations, and the detection of Taltirelin is limited to 2.42ng.

(3) chromatographic condition: be filling agent with octadecylsilane chemically bonded silica, the detection wavelength is 210nm, with the potassium dihydrogen phosphate buffer solution (15: 80) of acetonitrile-(0.01mol/L) as mobile phase, wherein containing concentration in potassium dihydrogen phosphate buffer solution is the 2g/1000mL sodium heptanesulfonate, and pH is 3.5.

By above-mentioned chromatographic condition, the Taltirelin reference substance solution is measured, with the peak area of each Taltirelin, sample size is carried out linear regression.Under this chromatographic system, Taltirelin is good in 0.25～5.01 μ g/ml concentration range internal linear relation, and the detection of Taltirelin is limited to 0.50ng.

2. the methodological study of impurity 1

By above-mentioned chromatographic condition, impurity 1 reference substance solution is measured, with the peak area of each impurity 1, sample size is carried out linear regression.Under this chromatographic system, impurity 1 is good in 0.83～8.50 μ g/ml concentration range internal linear relation, and the detection of impurity 1 is limited to 16.6ng.

By above-mentioned chromatographic condition, impurity 1 reference substance solution is measured, with the peak area of each impurity 1, sample size is carried out linear regression.Under this chromatographic system, impurity 1 is good in 0.55～6.42 μ g/ml concentration range internal linear relation, and the detection of impurity 1 is limited to 1.10ng.

(3) chromatographic condition: be filling agent with octadecylsilane chemically bonded silica, the detection wavelength is 210nm, with the potassium dihydrogen phosphate buffer solution (15: 80) of acetonitrile-(0.01mol/L) as mobile phase, wherein containing concentration in buffer solution is the 2g/1000mL sodium heptanesulfonate, and pH is 3.5.

By above-mentioned chromatographic condition, impurity 1 reference substance solution is measured, with the peak area of each impurity 1, sample size is carried out linear regression.Under this chromatographic system, impurity 1 is good in 0.25～4.98 μ g/ml concentration range internal linear relation, and the detection of impurity 1 is limited to 0.50ng.

3. the methodological study of impurity 2

By above-mentioned chromatographic condition, impurity 2 reference substance solution are measured, with the peak area of each impurity 2, sample size is carried out linear regression.Under this chromatographic system, impurity 2 is good in 0.78～7.22 μ g/ml concentration range internal linear relation, and the detection of impurity 2 is limited to 1.56ng.

By above-mentioned chromatographic condition, impurity 2 reference substance solution are measured, with the peak area of each impurity 2, sample size is carried out linear regression.Under this chromatographic system, impurity 2 is good in 0.35～4.51 μ g/ml concentration range internal linear relation, and the detection of impurity 2 is limited to 0.50ng.

By above-mentioned chromatographic condition, impurity 2 reference substance solution are measured, with the peak area of each impurity 2, sample size is carried out linear regression.Under this chromatographic system, impurity 2 is good in 0.25 μ g/ml-10.14 μ g/ml concentration range internal linear relation, and the detection of impurity 2 is limited to 0.51ngl.

4. the methodological study of impurity 3

By above-mentioned chromatographic condition, impurity 3 reference substance solution are measured, with the peak area of each impurity 3, sample size is carried out linear regression.Under this chromatographic system, impurity 3 is good in 1.32～9.01 μ g/ml concentration range internal linear relations, the detectability 2.64ng of impurity 3.

By above-mentioned chromatographic condition, impurity 3 reference substance solution are measured, with the peak area of each impurity 3, sample size is carried out linear regression.Under this chromatographic system, impurity 3 is good in 0.78～8.65 μ g/ml concentration range internal linear relation, and the detection of impurity 3 is limited to 1.56ng.

(3) chromatographic condition: be filling agent with octadecylsilane chemically bonded silica, the detection wavelength is 210nm, with the potassium dihydrogen phosphate buffer solution (15: 80) of acetonitrile-(0.01mol/L) as mobile phase, wherein containing concentration in buffer solution is the 2g/1000mL sodium heptanesulfonate, and pH is 2.5.

By above-mentioned chromatographic condition, impurity 3 reference substance solution are measured, with the peak area of each impurity 3, sample size is carried out linear regression.Under this chromatographic system, impurity 3 is good in 0.26 μ g/ml-10.26 μ g/ml concentration range internal linear relation, and the detection of impurity 3 is limited to 0.51ng/ml.

Description of drawings

Fig. 1 be the Taltirelin crude product at acetonitrile-water (30: 70) as the spectrogram under the condition of mobile phase;

Fig. 2 be the Taltirelin crude product in acetonitrile-0.10mol/L phosphate buffered solution (30: 70) as the spectrogram under the condition of mobile phase;

Fig. 3 be the Taltirelin crude product in acetonitrile-0.05mol/L phosphate buffered solution (10: 90) as the spectrogram under the condition of mobile phase;

Fig. 4 is that Taltirelin (is taking decane sodium sulfonate 1.0g with decane sodium sulfonate solution, add 0.01mol/L (phosphate total concentration) sodium dihydrogen phosphate, sodium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 1.0)-acetonitrile (80: 20) is as the spectrogram under the condition of mobile phase

Fig. 5 is that the Taltirelin crude product (is taking sodium heptanesulfonate 5.0g with the heptanesulfonic acid sodium solution, add 0.001mol/L (phosphate total concentration) sodium dihydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 5.0)-acetonitrile (80: 20) is as the spectrogram under the condition of mobile phase;

Fig. 6 be the Taltirelin sheet with heptanesulfonic acid sodium solution (take sodium heptanesulfonate 1.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (90: 10) as the spectrogram under the condition of mobile phase;

Fig. 7 be impurity 1 with heptanesulfonic acid sodium solution (take sodium heptanesulfonate 5.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (85: 15) as the spectrogram under the condition of mobile phase;

Fig. 8 be impurity 2 with perfluoroetane sulfonic acid sodium solution (take perfluorooctane sulfonate 4.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 2.5)-acetonitrile (90: 10) as the spectrogram under the condition of mobile phase;

Fig. 9 be impurity 3 with heptanesulfonic acid sodium solution (take sodium heptanesulfonate 5.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (90: 10) as the spectrogram under the condition of mobile phase;

Figure 10 be former grind Taltirelin with sodium hexanesulfonate solution (take sodium hexanesulfonate 4.0g, add 0.03mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (84: 16) as the spectrogram under the condition of mobile phase;

Figure 11 is that the former Taltirelin sheet specificity research aqueous solution failure test (3 hours) of grinding (is taking decane sodium sulfonate 2.0g with decane sodium sulfonate solution, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 2.5)-acetonitrile (85: 15) is as the spectrogram under the condition of mobile phase;

Figure 12 is that the former Taltirelin sheet specificity research aqueous solution failure test (10 days) of grinding (is taking sodium pentanesulfonate 6.0g with sodium pentanesulfonate solution, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3.0)-acetonitrile (85: 15) is as the spectrogram under the condition of mobile phase.

Embodiment

The present invention will be further described below by embodiment, but embodiment does not limit protection scope of the present invention.

Embodiment 1

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With the sodium dihydrogen phosphate buffer (30: 70) of acetonitrile-(0.001mol/L) as mobile phase; Detect wavelength 200nm; 30 ℃ of column temperatures.

The preparation of impurity reference substance solution: get respectively impurity 1, impurity 2, impurity 3 reference substances in volumetric flask, add mobile phase and be diluted to scale, shake up as the impurity contrast solution.

The preparation of need testing solution: precision takes the Taltirelin crude product and puts in right amount in volumetric flask, adds methyl alcohol dissolving, and adds mobile phase and be diluted to scale, shakes up, as need testing solution.

The preparation of contrast solution: precision measures need testing solution 1ml, puts in the 100ml measuring bottle, and mobile phase is diluted to scale, shakes up, in contrast solution.

Determination method: get respectively impurity reference substance solution, need testing solution, contrast solution injection liquid chromatography, and record chromatogram.

Testing result shows that between Taltirelin main peak, impurity peaks and solvent peak, degree of separation is better, but the peak type at each peak is bad, and is influential to the precision that detects.

Embodiment 2

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With the disodium hydrogen phosphate buffer solution (20: 80) of acetonitrile-(0.05mol/L) as mobile phase; Detect wavelength 205nm; 30 ℃ of column temperatures.

The preparation of impurity reference substance solution: get respectively impurity 1, impurity 2, impurity 3 reference substances in measuring bottle, add mobile phase and be diluted to scale, shake up as the impurity contrast solution.

Embodiment 3

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With the potassium dihydrogen phosphate buffer solution (10: 90) of acetonitrile-(0.1mol/L) as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Embodiment 4

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With the dipotassium hydrogen phosphate buffer solution (15: 85) of acetonitrile-(0.01mol/L) as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Embodiment 5

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With acetonitrile-(0.01mol/L (phosphate total concentration)) sodium hydrogen phosphate, sodium hydrogen phosphate mixing buffer solution (20: 80) as mobile phase; Detect wavelength 200nm; 30 ℃ of column temperatures.

Embodiment 6

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With acetonitrile-(0.1mol/L (phosphate total concentration)) dipotassium hydrogen phosphate, dipotassium hydrogen phosphate mixing buffer solution (30: 70) as mobile phase; Detect wavelength 205nm; 30 ℃ of column temperatures.

Determination method: get respectively impurity reference substance solution, need testing solution, contrast solution injection liquid chromatography, and record chromatogram (the test sample chromatogram is seen accompanying drawing 2).

Embodiment 7

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With acetonitrile-(0.05mol/L (phosphate total concentration)) sodium hydrogen phosphate, dipotassium hydrogen phosphate mixing buffer solution (10: 90) as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Determination method: get respectively impurity reference substance solution, need testing solution, contrast solution injection liquid chromatography, and record chromatogram (the test sample chromatogram is seen accompanying drawing 3).

Embodiment 8

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With acetonitrile-(0.001mol/L (phosphate total concentration)) sodium dihydrogen phosphate, dipotassium hydrogen phosphate mixing buffer solution (20: 80) as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Embodiment 9

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With acetonitrile-(0.05mol/L (phosphate total concentration)) sodium dihydrogen phosphate, potassium dihydrogen phosphate mixing buffer solution (30: 70) as mobile phase; Detect wavelength 200nm; 30 ℃ of column temperatures.

Embodiment 10

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With acetonitrile-(0.01mol/L (phosphate total concentration)) sodium hydrogen phosphate, dipotassium hydrogen phosphate mixing buffer solution (30: 70) as mobile phase; Detect wavelength 205nm; 30 ℃ of column temperatures.

Embodiment 11

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With the disodium hydrogen phosphate buffer solution (10: 90) of acetonitrile-(0.01mol/L) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

Embodiment 12

Take self-control Taltirelin crude product as raw material

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; (take sodium pentanesulfonate 2.0g, add 0.001mol/L sodium dihydrogen phosphate 1000ml and make dissolving, with phosphoric acid adjust pH to 1)-acetonitrile (70: 30) is mobile phase with sodium pentanesulfonate solution; The detection wavelength is 200nm; 30 ℃ of column temperatures.

Testing result shows that between Taltirelin main peak, impurity peaks and solvent peak, degree of separation is better, and the peak type is better, can accurately detect major component and each impurity component content in the Taltirelin crude product.

Embodiment 13

Take self-control Taltirelin crude product as raw material

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; (take sodium hexanesulfonate 5.0g, add 0.05mol/L sodium dihydrogen phosphate 1000ml and make dissolving, with phosphoric acid adjust pH to 5)-acetonitrile (80: 20) is mobile phase with sodium hexanesulfonate solution; The detection wavelength is 200nm; 30 ℃ of column temperatures.

Embodiment 14

Take self-control Taltirelin crude product as raw material

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; (take sodium heptanesulfonate 8.0g, add 0.1mol/L potassium dihydrogen phosphate 1000ml and make dissolving, with phosphoric acid adjust pH to 4.5)-acetonitrile (90: 10) is mobile phase with the heptanesulfonic acid sodium solution; The detection wavelength is 210nm; 30 ℃ of column temperatures.

Embodiment 15

Take self-control Taltirelin crude product as raw material

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; (take perfluorooctane sulfonate 10.0g, add 0.01mol/L dipotassium hydrogen phosphate solution 1000ml and make dissolving, with phosphoric acid adjust pH to 2.5)-acetonitrile (85: 15) is mobile phase with the perfluoroetane sulfonic acid sodium solution; The detection wavelength is 210nm; 30 ℃ of column temperatures.

Embodiment 16

Take self-control Taltirelin elaboration as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take decane sodium sulfonate 1.0g with decane sodium sulfonate solution, add 0.01mol/L (phosphate total concentration) sodium dihydrogen phosphate, sodium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 1)-acetonitrile (80: 20) is as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Determination method: get respectively impurity reference substance solution, need testing solution, contrast solution injection liquid chromatography, and record chromatogram (the test sample spectrogram is seen accompanying drawing 4).

Embodiment 17

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take sodium pentanesulfonate 6.0g with sodium pentanesulfonate solution, add 0.1mol/L (phosphate total concentration) potassium dihydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 2.5)-acetonitrile (80: 20) is as mobile phase; Detect wavelength 205nm; 30 ℃ of column temperatures.

Embodiment 18

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take sodium hexanesulfonate 7.0g with sodium hexanesulfonate solution, add 0.05mol/L (phosphate total concentration) sodium dihydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 4.0)-acetonitrile (90: 10) is as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Embodiment 19

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take sodium heptanesulfonate 4.0g with the heptanesulfonic acid sodium solution, add 0.001mol/L (phosphate total concentration) sodium dihydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 5.0)-acetonitrile (80: 20) is as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Determination method: get respectively impurity reference substance solution, need testing solution, contrast solution injection liquid chromatography, and record chromatogram (the test sample chromatogram is seen accompanying drawing 5).

Embodiment 20

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take perfluorooctane sulfonate 6.0g with the perfluoroetane sulfonic acid sodium solution, add 0.05mol/L (phosphate total concentration) sodium dihydrogen phosphate, potassium dihydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 1.0)-acetonitrile (70: 30) is as mobile phase; Detect wavelength 200nm; 30 ℃ of column temperatures.

Embodiment 21

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take decane sodium sulfonate 10.0g with decane sodium sulfonate solution, add 0.01mol/L (phosphate total concentration) sodium hydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 2.5)-acetonitrile (70: 30) is as mobile phase; Detect wavelength 205nm; 30 ℃ of column temperatures.

Embodiment 22

Take self-control Taltirelin crude product as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With heptanesulfonic acid sodium solution (take sodium heptanesulfonate 8.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (90: 10) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

Embodiment 23

Take self-control Taltirelin sheet as raw material

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; (take perfluorooctane sulfonate 8.0g, add 0.001mol/L sodium dihydrogen phosphate 1000ml and make dissolving, with phosphoric acid adjust pH to 5)-acetonitrile (70: 30) is mobile phase with the perfluoroetane sulfonic acid sodium solution; The detection wavelength is 200nm; 30 ℃ of column temperatures.

The preparation of need testing solution: get the content under Taltirelin sheet loading amount difference item, mix, take in right amount and put in volumetric flask, add methyl alcohol dissolving, and add mobile phase and be diluted to scale, shake up, as need testing solution.

Testing result shows that between Taltirelin main peak, impurity peaks and solvent peak, degree of separation is better, and the peak type is better, can accurately detect major component and each impurity component content in the Taltirelin sheet.

Embodiment 24

Take self-control Taltirelin sheet as raw material

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; (take decane sodium sulfonate 2.0g, add 0.05mol/L sodium dihydrogen phosphate 1000ml and make dissolving, with phosphoric acid adjust pH to 1)-acetonitrile (80: 20) is mobile phase with decane sodium sulfonate solution; The detection wavelength is 205nm; 30 ℃ of column temperatures.

Embodiment 25

Take self-control Taltirelin sheet as raw material

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; (take sodium pentanesulfonate 1.0g, add 0.1mol/L potassium dihydrogen phosphate 1000ml and make dissolving, with phosphoric acid adjust pH to 3.5)-acetonitrile (90: 10) is mobile phase with sodium pentanesulfonate solution; The detection wavelength is 210nm; 30 ℃ of column temperatures.

Embodiment 26

Take self-control Taltirelin sheet as raw material

Chromatographic condition: be filling agent with octadecylsilane chemically bonded silica; (take sodium heptanesulfonate 4.0g, add 0.01mol/L dipotassium hydrogen phosphate solution 1000ml and make dissolving, with phosphoric acid adjust pH to 2.5)-acetonitrile (85: 15) is mobile phase with the heptanesulfonic acid sodium solution; The detection wavelength is 210nm; 30 ℃ of column temperatures.

Embodiment 27

Take self-control Taltirelin sheet as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take sodium hexanesulfonate 10.0g with sodium hexanesulfonate solution, add 0.01mol/L (phosphate total concentration) sodium dihydrogen phosphate, sodium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 5)-acetonitrile (80: 20) is as mobile phase; Detect wavelength 200nm; 30 ℃ of column temperatures.

Embodiment 28

Take self-control Taltirelin sheet as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take decane sodium sulfonate 2.0g with decane sodium sulfonate solution, add 0.1mol/L (phosphate total concentration) potassium dihydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 1.0)-acetonitrile (70: 30) is as mobile phase; Detect wavelength 205nm; 30 ℃ of column temperatures.

Embodiment 29

Take self-control Taltirelin sheet as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take sodium pentanesulfonate 6.0g with sodium pentanesulfonate solution, add 0.05mol/L (phosphate total concentration) sodium dihydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 1.5)-acetonitrile (90: 10) is as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Embodiment 30

Take self-control Taltirelin sheet as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take sodium hexanesulfonate 10.0g with sodium hexanesulfonate solution, add 0.001mol/L (phosphate total concentration) sodium dihydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 1.0)-acetonitrile (80: 20) is as mobile phase; Detect wavelength 210nm; 30 ℃ of column temperatures.

Embodiment 31

Take self-control Taltirelin sheet as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take sodium heptanesulfonate 5.0g with the heptanesulfonic acid sodium solution, add 0.001mol/L (phosphate total concentration) sodium dihydrogen phosphate, potassium dihydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 4.5)-acetonitrile (70: 30) is as mobile phase; Detect wavelength 200nm; 30 ℃ of column temperatures.

Embodiment 32

Take self-control Taltirelin sheet as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; (take perfluorooctane sulfonate 4.0g with the perfluoroetane sulfonic acid sodium solution, add 0.01mol/L (phosphate total concentration) sodium hydrogen phosphate, dipotassium hydrogen phosphate mixed solution 1000ml makes dissolving, with phosphoric acid adjust pH to 3.0)-acetonitrile (70: 30) is as mobile phase; Detect wavelength 205nm; 30 ℃ of column temperatures.

Embodiment 33

Take self-control Taltirelin sheet as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With heptanesulfonic acid sodium solution (take sodium heptanesulfonate 1.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (90: 10) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

Determination method: get respectively impurity reference substance solution, need testing solution, contrast solution injection liquid chromatography, and record chromatogram.(the test sample chromatogram is seen accompanying drawing 6)

Embodiment 34

Take impurity 1 as product to be tested.

Chromatographic condition is: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With heptanesulfonic acid sodium solution (take sodium heptanesulfonate 5.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (85: 15) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

Precision takes impurity 1 and puts in volumetric flask, adds mobile phase and is diluted to scale, shakes up, and the accurate test sample injection liquid chromatography of drawing records chromatogram (seeing accompanying drawing 7).

Conclusion: by spectrogram as can be known, retention time 3.08 minutes, the peak type is better.

Embodiment 35

Take impurity 2 as product to be tested.

Chromatographic condition is: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With perfluoroetane sulfonic acid sodium solution (take perfluorooctane sulfonate 4.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 2.5)-acetonitrile (90: 10) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

Precision takes impurity 2 and puts in volumetric flask, adds mobile phase and is diluted to scale, shakes up, and the accurate test sample injection liquid chromatography of drawing records chromatogram (seeing accompanying drawing 8).

Conclusion: by spectrogram as can be known, retention time 14.353 minutes, the peak type is better.

Embodiment 36

Take impurity 3 as product to be tested.

Chromatographic condition is: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With heptanesulfonic acid sodium solution (take sodium heptanesulfonate 5.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (90: 10) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

Precision takes impurity 1 and puts in volumetric flask, adds mobile phase and is diluted to scale, shakes up, and the accurate test sample injection liquid chromatography of drawing records chromatogram (seeing accompanying drawing 9).

Conclusion: by spectrogram as can be known, retention time 16.812 minutes, the peak type is better.

Embodiment 37

Take the former Taltirelin sheet that grinds as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With sodium hexanesulfonate solution (take sodium hexanesulfonate 4.0g, add 0.03mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3)-acetonitrile (84: 16) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

The preparation of need testing solution: get the content under Taltirelin sheet loading amount difference item, mix, take in right amount and put in volumetric flask, add mobile phase and be diluted to scale, shake up, as need testing solution.

Determination method: get respectively impurity reference substance solution, need testing solution, contrast solution injection liquid chromatography, and record chromatogram.(the test sample chromatogram is seen accompanying drawing 10)

Embodiment 38 specificity research aqueous solution failure tests (3 hours)

Take the former Taltirelin sheet that grinds as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With decane sodium sulfonate solution (take decane sodium sulfonate 2.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 2.5)-acetonitrile (85: 15) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

The preparation of need testing solution: get the content under Taltirelin sheet loading amount difference item, mix, take in right amount and put in volumetric flask, add mobile phase and be diluted to scale, shake up, put in 80 ℃ of water-baths constant temperature 3 hours, filter, get subsequent filtrate as need testing solution.

Get need testing solution injection liquid chromatography, and record chromatogram.(the test sample chromatogram is seen accompanying drawing 11)

Testing result shows: be that decon 2,12.80 is the increase in this test of impurity 3, impurity 1 in the main peak front peak that went out in 10.70 minutes.Result shows that this product constant temperature 2 hours in 80 ℃ of water-baths of water-bath is unstable.

Embodiment 38 specificity research aqueous solution failure tests (10 days)

Take the former Taltirelin sheet that grinds as raw material

Chromatographic condition: octadecylsilane chemically bonded silica is the chromatographic column of filling agent; With sodium pentanesulfonate solution (take sodium pentanesulfonate 6.0g, add 0.01mol/L disodium phosphate soln 1000ml and make dissolving, with phosphoric acid adjust pH to 3.0)-acetonitrile (85: 15) as mobile phase; Detect wavelength 210m; 30 ℃ of column temperatures.

The preparation of need testing solution: get the content under Taltirelin sheet loading amount difference item, mix, take in right amount and put in volumetric flask, add mobile phase and be diluted to scale, shake up, put in 80 ℃ of water-baths constant temperature 10 days, filter, get subsequent filtrate as need testing solution.

Get need testing solution injection liquid chromatography, and record chromatogram.(the test sample chromatogram is seen accompanying drawing 12)

Testing result shows: in the main peak front peak that went out in 10.271 minutes be decon 2, with main peak before 12.136 be the increase in this test of impurity 3, impurity 1.Result shows that this product constant temperature 10 days in 80 ℃ of water-baths of water-bath is unstable.

Claims

1. a Taltirelin and the assay of preparation and the high performance liquid chromatography of impurity determination, it is characterized in that octadecylsilane chemically bonded silica is the chromatographic column of filling agent, so that acetonitrile-(phosphate buffered solution (30: 70～10: 90) of 0.001mol/L～0.10mol/L) is as mobile phase.

2. method according to claim 1, the ratio that it is characterized in that described acetonitrile-phosphate buffered solution is 15: 85.

3. method according to claim 1, the concentration that it is characterized in that described phosphate buffered solution is 0.01mol/L.

4. according to claim 1,2,3 described methods, it is characterized in that described phosphate is selected from one or more in sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate or dipotassium hydrogen phosphate.

5. according to claim 1,2,3 described methods, it is characterized in that containing in phosphate buffered solution the ion-pairing agent that concentration is 1～10g/1000mL.

6. method according to claim 5, is characterized in that described ion-pairing agent is selected from one or more in sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, perfluorooctane sulfonate, decane sodium sulfonate.

7. method according to claim 6, the pH value that it is characterized in that containing the phosphate buffered solution of ion-pairing agent is 1～5.

8. method according to claim 7, is characterized in that the pH value is 2.5.