CN107664666A

CN107664666A - A kind of relevant substance-measuring method of cetrorelix acetate and its preparation

Info

Publication number: CN107664666A
Application number: CN201710869282.2A
Authority: CN
Inventors: 崔学云; 赵晓亮; 潘正斐; 邓德雄
Original assignee: HAINAN ZHONGHE PHARMACEUTICAL CO Ltd
Current assignee: HAINAN ZHONGHE PHARMACEUTICAL CO Ltd
Priority date: 2017-09-23
Filing date: 2017-09-23
Publication date: 2018-02-06
Anticipated expiration: 2037-09-23
Also published as: CN107664666B

Abstract

The invention discloses a kind of relevant substance-measuring method of cetrorelix acetate and its preparation, methods described step are as follows：Step 1, Cetrorelix impurity is weighed, adds acetonitrile to dissolve, then is diluted with acetonitrile and sulfuric acid salt buffer solution, impurity reference substance solution is made；Step 2, Cetrorelix is weighed, adds acetonitrile to make dissolving, then is diluted with acetonitrile and sulfuric acid salt buffer solution, is configured to the solution containing Cetrorelix；Step 3, take impurity reference substance solution and need testing solution to be injected separately into liquid chromatograph, record chromatogram, calculate the content of each impurity respectively with peak area by external standard method；This method efficiently solve separation and its preparation relevant material the problem of, this method specificity is strong, reproducible, can guarantee that and its preparation in the controllability about material.

Description

A kind of relevant substance-measuring method of cetrorelix acetate and its preparation

Technical field

The present invention relates to the analysis method method of inspection of a kind of active peptides raw material medicine and preparation, more particularly to acetic acid west The method of the relevant substance-measuring of Qu Ruike and its preparation.

Background technology：

Cetrorelix acetate (Cetrorelix Acetate), its chemical history is referred to as：N- acetyl -3- (2- naphthalenes) third ammonia of-D- Acyl-p- chloro- D- phenylalanyls -3- (3- pyridine radicals)-D- alanyl-L- seryl-L- tyrosyl-N5- carbamyl-D- bird ammonia Acyl-L- leucyl-L- arginyl-L- prolyl-D- alanimamides acetate.

Its structural formula：

Cetrorelix acetate is a kind of gonadotropin-releasing hormone (GRH) (GnRH) antagonist.On GnRH and pituicyte film Acceptor combines, and stimulates lutropin (LH) and follicle-stimulating hormone (FSH) (FSH) release.Cetrorelix acetate and endogenous GnRH The acceptor on pituicyte is competed, so as to suppress the release of endogenous LH and FSH.The inhibitory action of cetrorelix acetate is being injected Produce immediately afterwards, without stimulation.In women, the effect of cetrorelix acetate is to postpone the appearance at LH peaks, so as to the row of control Ovum.

Due to synthetic method, plurality of impurities can be mingled with cetrorelix acetate bulk drug, these impurity include this Following impurity prepared by inventor：

These impurity can more or less influence the quality of cetrorelix acetate product, it is therefore necessary to carry out content control to it System, therefore, the present invention constructs a kind of analysis method of Cetrorelix about material, this method answers specificity good, and energy will likely Degradation impurity and possible residual intermediate effectively separated with principal component, and high sensitivity can be applied to the matter of Cetrorelix Amount control.

The content of the invention：

The present invention provides a kind of cetrorelix acetate and its detection method of the preparation about material, and methods described step is such as Under：

Step 1, reference substance solution is prepared：

Cetrorelix impurity is weighed, adds acetonitrile to dissolve, then is diluted with acetonitrile and sulfuric acid salt buffer solution, impurity control is made Product solution；

Step 2, need testing solution is prepared：

Cetrorelix is weighed, adds acetonitrile to make dissolving, then is diluted with acetonitrile and sulfuric acid salt buffer solution, is configured to contain western Qu Rui Gram solution；

Step 3, determine

Take impurity reference substance solution and need testing solution be injected separately into liquid chromatograph, record chromatogram, by external standard method with Peak area calculates the content of each impurity respectively；

Wherein, the chromatographic condition of the high performance liquid chromatography is as follows：

Using octadecylsilane chemically bonded silica as chromatographic column filler, using UV-detector, delayed from acetonitrile and sulfate It is mobile phase to rush solution, gradient elution；Wherein described gradient elution, mode are as follows：

0~10 minute, acetonitrile elution ratio 0~20%, 10~30 minutes, acetonitrile elution ratio 10%~40%, 30~ 50 minutes, acetonitrile elution ratio 30%~60%, 50~70 minutes, acetonitrile elution ratio was maintained at 30%~60%%；Detection Wavelength is 210nm~240nm；Flow velocity is 0.8~1.5ml/min；Sample size is 5~50 μ l.

Wherein described sulfate cushioning liquid is selected from：Ammonium sulfate, metabisulfite solution, potassium sulfate solution, or sulfuric acid and The mixed solution of ammoniacal liquor, buffer concentration are no more than 0.1%, and pH value is 2~3.

Preferably, detection method of the present invention, step are as follows：

Step 1, reference substance solution is prepared：

It is appropriate that precision weighs Cetrorelix impurity, adds a small amount of acetonitrile to dissolve, then with volume ratio 2:5 acetonitrile delays with sulfate Rush solution dilution and the respectively solution containing 0.005mg is made in every 1ml, as impurity reference substance solution；

Step 2, need testing solution is prepared：

Cetrorelix is weighed, adds a small amount of acetonitrile to make dissolving, then with volume ratio 2:5 acetonitrile dilutes with sulfuric acid salt buffer solution It is configured to the solution that concentration containing Cetrorelix is 0.5mg/ml；

Step 3, determine

Precision measures impurity reference substance solution and need testing solution is injected separately into liquid chromatograph, chromatogram is recorded, by outer Mark method calculates the content of each impurity with peak area respectively.

The method of the present invention is obtained by screening, and screening process is as follows：

2.4.2 the screening of mobile phase species：Phosphate-buffered liquid system and sulfate buffer system is selected to carry out west song Groping for the detection method that may include impurity in Rake main peak, is as a result shown in Fig. 1-Fig. 2.

Fig. 1 results are shown：Sulfate system is bright for the separation under the segregation ratio phosphate system of other impurities before main peak Show more, so selection sulfate system.

Fig. 2 results are shown：In phosphate system, each known impurities and main peak can be completely separable, but indivedual known impurities Between fail to be kept completely separate；In sulfate system, each known impurities and main peak can be kept completely separate, and effect becomes apparent from, so Select sulfate system.

2.4.3 the selection of gradient

2.4.3.1 use high performance liquid chromatography, using octadecylsilane chemically bonded silica as filler (4.6 × 250mm, 4.6 μm), mobile phase is 0.5% sulfate buffer (A)-acetonitrile (B)；Flow velocity is 1.0mL/min, Detection wavelength 226nm；Enter The μ l of sample amount 20；Condition of gradient elution is shown in Table 1.

The gradient elution table of table 1

The chromatogram obtained under the conditions of this is shown in Fig. 3, and other impurities are wrapped in main peak before main peak.

2.4.3.2 the same 2.4.3.1 of condition, condition of gradient elution are shown in Table 2.

Time (min)	Mobile phase A %	Mobile phase B %
			0	85	15
10	80	20
			25	76	24
30	72	28
			45	60	40
50	60	40
			51	85	15
60	85	15

The chromatogram obtained under the conditions of this is shown in Fig. 4, and other impurities separation makes moderate progress before main peak, but not up to separates.

2.4.3.3 the same 2.4.3.1 of condition, condition of gradient elution are shown in Table 3.

The gradient elution table of table 3

Time (min)	Mobile phase A %	Mobile phase B %
			0	83	17
35	62	38
			55	55	45
65	55	45
			66	83	17
75	83	17

The chromatogram obtained under the conditions of this is shown in Fig. 5, and other impurities peak separating effect is obvious before main peak, and separating degree is more than 1.5.

2.4.4 the selection of column temperature

According to the gradient under the chromatographic condition and 2.4.3.3 under 2.4.3.1, select respectively 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C of column temperatures are tested.As a result Fig. 6-Figure 10 is seen.

Fig. 6-Figure 10 results show that 45 DEG C, 50 DEG C and 55 DEG C of chromatogram peak type is preferable, and can be isolated before main peak two it is miscellaneous Matter, separating effect are obvious, it is contemplated that pillar service life problem, it is advantageous to 45 DEG C of column temperatures.

Brief description of the drawings

Fig. 1-1：Phosphate buffer

Fig. 1-2：Sulfate buffer

Fig. 2-1：Known impurities separation situation under phosphate-buffered liquid system

Fig. 2-2：Known impurities separation situation under sulfate buffer system

Fig. 3：Impurity separates situation under the system of gradient 1

Fig. 4：Impurity separates situation under the system of gradient 2

Fig. 5：Impurity separates situation under the system of gradient 3

Fig. 6：Impurity separates situation at 35 DEG C of column temperature

Fig. 7：Impurity separates situation at 40 DEG C of column temperature

Fig. 8：Impurity separates situation at 45 DEG C of column temperature

Fig. 9：Impurity separates situation at 50 DEG C of column temperature

Figure 10：Impurity separates situation at 55 DEG C of column temperature

Embodiment：

The present invention is further illustrated by the following examples, but not as limitation of the present invention.

Embodiment 1

A kind of detection method of cetrorelix acetate and its preparation about material, methods described step are as follows：

Step 1, reference substance solution is prepared：

It is appropriate that precision weighs each impurity of Cetrorelix, adds a small amount of acetonitrile to dissolve, then dilute with buffering mixed salt solution with acetonitrile Release (2:5) the respectively solution containing 0.005mg is made in every 1ml, as impurity reference substance solution.

Step 2, need testing solution is prepared：

Cetrorelix is weighed, adds a small amount of acetonitrile to make dissolving, then diluted with acetonitrile and buffering mixed salt solution, the mixed solution In, the ratio of acetonitrile is 20:50, it is configured to the solution that concentration containing Cetrorelix is 0.5mg/ml.

Step 3, determine

By standard reference material solution and need testing solution injection high performance liquid chromatograph, chromatogram is obtained, according to chromatogram, Calculate the content of known impurities in sample.

Wherein, the high performance liquid chromatography, chromatographic condition are as follows：

Using octadecylsilane chemically bonded silica as chromatographic column filler, using UV-detector, delayed from acetonitrile and sulfate It is mobile phase to rush solution, gradient elution；Wherein described sulfate cushioning liquid includes but is not limited to sulfuric acid solution, and ammonium sulfate is molten Liquid, metabisulfite solution and potassium sulfate solution, such as use 0.5%H₂SO₄Solution, or ammoniacal liquor regulation PH to 2~3；

Wherein described gradient elution, mode are as follows：

0~10 minute, acetonitrile elution ratio 0~20%, 10~30 minutes, acetonitrile elution ratio 10%~40%, 30~ 50 minutes, acetonitrile elution ratio 30%~60%, 50~70 minutes, acetonitrile elution ratio was maintained at 30%~60%%；Detection Wavelength is 210nm~240nm；Flow velocity is 0.8~1.5ml/min；Sample size is 5~50 μ l；Buffer concentration is no more than 0.1%, pH value is 2~3.

Claims

A kind of 1. detection method of cetrorelix acetate and its preparation about material, it is characterised in that：Step is as follows：

Step 1, reference substance solution is prepared：

Cetrorelix impurity is weighed, adds acetonitrile to dissolve, then is diluted with acetonitrile and sulfuric acid salt buffer solution, it is molten that impurity reference substance is made Liquid；

Step 2, need testing solution is prepared：

Cetrorelix is weighed, adds acetonitrile to make dissolving, then is diluted with acetonitrile and sulfuric acid salt buffer solution, is configured to containing Cetrorelix Solution；

Step 3, determine

Take impurity reference substance solution and need testing solution to be injected separately into liquid chromatograph, chromatogram is recorded, by external standard method with peak face Integration does not calculate the content of each impurity；

Wherein, the chromatographic condition of the high performance liquid chromatography is as follows：

It is molten from acetonitrile and sulfuric acid salt buffer using UV-detector using octadecylsilane chemically bonded silica as chromatographic column filler Liquid is mobile phase, gradient elution；Wherein described gradient elution, mode are as follows：

0~10 minute, acetonitrile elution ratio 0~20%, 10~30 minutes, acetonitrile elution ratio 10%~40%, 30~50 points Clock, acetonitrile elution ratio 30%~60%, 50~70 minutes, acetonitrile elution ratio was maintained at 30%~60%%；Detection wavelength For 210nm~240nm；Flow velocity is 0.8~1.5ml/min；Sample size is 5~50 μ l.
2. detection method according to claim 1, it is characterised in that：

Wherein described sulfate cushioning liquid is selected from：Ammonium sulfate, metabisulfite solution, potassium sulfate solution, or sulfuric acid and ammoniacal liquor Mixed solution, buffer concentration be no more than 0.1%, pH value be 2~3.
3. detection method according to claim 1, it is characterised in that：Step is as follows：

Step 1, reference substance solution is prepared：

It is appropriate that precision weighs Cetrorelix impurity, adds a small amount of acetonitrile to dissolve, then with volume ratio 2:5 acetonitrile and sulfuric acid salt buffer are molten The respectively solution containing 0.005mg is made in every 1ml in liquid dilution, as impurity reference substance solution；

Step 2, need testing solution is prepared：

Cetrorelix is weighed, adds a small amount of acetonitrile to make dissolving, then with volume ratio 2:5 acetonitrile is prepared with the dilution of sulfuric acid salt buffer solution Into the solution that concentration containing Cetrorelix is 0.5mg/ml；

Step 3, determine

Precision measures impurity reference substance solution and need testing solution is injected separately into liquid chromatograph, chromatogram is recorded, by external standard method Calculate the content of each impurity respectively with peak area.
4. detection method according to claim 1, it is characterised in that：The impurity is：