CN109655540B - Method for analyzing related substances of salmon calcitonin nasal spray - Google Patents

Method for analyzing related substances of salmon calcitonin nasal spray Download PDF

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CN109655540B
CN109655540B CN201811563528.4A CN201811563528A CN109655540B CN 109655540 B CN109655540 B CN 109655540B CN 201811563528 A CN201811563528 A CN 201811563528A CN 109655540 B CN109655540 B CN 109655540B
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salmon calcitonin
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calcitonin
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CN109655540A (en
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潘缘媛
李杨君
倪庆元
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YinGu Pharmaceutical Co Ltd
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Abstract

The invention discloses an analysis method of salmon calcitonin nasal spray related substances, which adopts high performance liquid chromatography to analyze and measure the salmon calcitonin nasal spray related substances. The method is verified by methodology, has strong specificity, high sensitivity, good repeatability and high accuracy, and can effectively control relevant substances of preparations such as salmon calcitonin, salmon calcitonin nasal spray and the like.

Description

Method for analyzing related substances of salmon calcitonin nasal spray
Technical Field
The invention belongs to the technical field of medicines, and relates to a salmon calcitonin and a detection method of related substances in a preparation thereof.
Background
Salmon calcitonin, also known as calcitonin, British name Calcitonin (Salmon), and molecular formula C 145 H 240 N 44 O 4 8S 2 Molecular weight 3431.89, CAS number: 47931-85-1. The structural formula is as follows:
H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH 2
the production process of salmon calcitonin mainly adopts a solid-phase synthesis method, namely fixing the C end of amino acid on insoluble resin, then condensing the amino acid on the resin in sequence, repeating the operations of condensation, washing, deprotection, neutralization washing and next round of condensation to reach the length of a peptide chain to be synthesized, finally cracking the peptide chain from the resin, and purifying to obtain the salmon calcitonin. Therefore, many impurities such as a polypeptide lacking one amino acid, a polypeptide lacking two amino acids, isomers resulting from racemization of amino acids, a linear polypeptide in which disulfide bonds are not formed, a hydrolyzed peptide in which asparagine or glutamine is hydrolyzed to aspartic acid or glutamic acid, and the like are produced. Which contains five impurities and has the following structural formula:
Figure BDA0001913925830000011
impurity A: r 1 ＝CO-CH 3 ，R 2 ＝NH 2 X-Leu; acetyl salmon calcitonin;
impurity B: r 1 ＝H，R 2 ＝NH 2 X-Leu; [ 9-D-leucine]-salmon calcitonin;
Figure BDA0001913925830000012
impurity C: des-22-tyrosine salmon calcitonin
Figure BDA0001913925830000013
Impurity D: oxyacetylated salmon calcitonin
Figure BDA0001913925830000021
Calcitonin C
The nasal spray standard of USP37 salmon calcitonin is adopted, and the salmon calcitonin raw material BP2014/EP8.3 and the Chinese pharmacopoeia method are difficult to detect due to more impurities and poor impurity separation degree.
The salmon calcitonin is a polypeptide which is chemically synthesized and consists of thirty-two amino acids, has the same structure as natural salmon calcitonin I, is degraded to a certain degree under the conditions of acid, alkali, oxidation, high temperature and illumination, and most impurities are generated by degradation, so that the conventional method is difficult to effectively detect, influences the quality control of the salmon calcitonin nasal spray to a certain degree, and also needs to establish an effective analysis method to strictly control.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting salmon calcitonin and related substances in the preparation thereof by using high performance liquid chromatography, which can effectively analyze and quantitatively determine impurities with ultraviolet absorption in the salmon calcitonin, thereby effectively controlling the quality of salmon calcitonin nasal spray products, and has the advantages of strong specificity, high sensitivity and good accuracy.
The invention adopts the following technical scheme:
a method for detecting salmon calcitonin and related substances in preparation thereof adopts high performance liquid chromatography for detection, and HPLC chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a stationary phase; the mobile phase A is a mixed solution of an inorganic salt buffer solution and an organic solvent; the mobile phase B is a mixed solution of inorganic salt buffer solution and organic solvent; the gradient elution conditions were as follows:
time/minute Mobile phase A/%) Mobile phase B/%)
0 70-75 30-25
30 55-60 45-40
32 70-75 30-25
55 70-75 30-25
Or the gradient elution conditions were as follows:
Figure BDA0001913925830000022
Figure BDA0001913925830000031
preferably, the inorganic salt in the inorganic salt buffer is one or more of tetramethylammonium hydroxide, ammonium formate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate, and more preferably tetrabutylammonium hydroxide.
Preferably, the concentration of the inorganic salt in the inorganic salt buffer is 0.01-0.05mol/L, preferably 0.01-0.03mol/L, more preferably 0.01-0.02mol/L, and most preferably 0.02 mol/L; and/or the pH value of the inorganic salt buffer ranges from 2.0 to 5.0, preferably from 2.0 to 4.0, most preferably 2.5.
Preferably, the organic solvent is one or more of methanol, acetonitrile and isopropanol, more preferably methanol and or acetonitrile.
Preferably, the mobile phase A is a mixed solution of 0.02mol/L tetramethylammonium hydroxide solution and acetonitrile, the volume ratio of the two is 9:1, wherein the tetramethylammonium hydroxide solution is adjusted to pH 2.5 by phosphoric acid;
the mobile phase B is a mixed solution of 0.02mol/L tetramethylammonium hydroxide solution and acetonitrile, the volume ratio of the two is 2:3, wherein the pH of the tetramethylammonium hydroxide solution is adjusted to 2.5 by phosphoric acid.
Preferably, the gradient elution conditions are as follows:
time per minute Mobile phase A/%) Mobile phase B/%)
0 70-90 30-10
30 60-40 40-60
32 60-100 40-0
55 60-100 40-0
More preferably, the gradient elution conditions are as follows:
time/minute Mobile phase A/%) Mobile phase B/%)
0 72 28
30 55 45
32 72 28
55 72 28
Preferably, the column temperature used is from 25 to 70 deg.C, preferably from 35 to 70 deg.C, more preferably from 50 to 70 deg.C, even more preferably from 60 to 65 deg.C, and most preferably 62 deg.C.
The stationary phase used may be C18 (4.6X 250mm, 5 μm) as column.
In particular, the column used may be Vydac 218TP C18,4.6 x 250mm,
Figure BDA0001913925830000041
5 μm, more preferably ZORBAX 300SB-C18, 4.6X 250mm, 5 μm.
Preferably, the flow rate of the mobile phase is 0.8-1.2mL/min, preferably 0.9-1.1mL/min, more preferably 1.0 mL/min.
Preferably, the sample is taken in an amount of 5-30. mu.l, preferably 10-30. mu.l, more preferably 20. mu.l.
Preferably, the detection wavelength is 210-254nm, more preferably 220 nm.
Furthermore, the salmon calcitonin or its preparation as the sample to be tested can be diluted to obtain a solution of the sample to be tested, preferably the concentration of the salmon calcitonin is 10-35 μ g/mL.
The diluent may be any one known to those skilled in the art, and is not particularly limited. In the invention, the preferable diluent is one or more of water, methanol and acetonitrile, and more preferably inorganic salt buffer solution-acetonitrile.
Preferably, the diluent is mobile phase A.
Furthermore, the salmon calcitonin nasal spray can be directly used as a sample solution to be detected.
The invention can adopt an ultraviolet detector for quantitative detection.
The impurity calcitonin C is a main degradation product of the calcitonin nasal spray, is easy to generate in solution and is not easy to appear in raw materials; the measurement results showed that the impurities were not detected after the starting material was left for two years. Therefore, the limit of the impurity is not controlled independently in the traditional Chinese medicine in domestic and foreign standards, but is controlled to be 7.0 percent in the injection. The impurity A is the only synthetic impurity detected from four known impurities (the impurities A, B, C and D), and the limit of the impurity A is not separately specified in salmon calcitonin nasal spray products at home and abroad. In order to strictly control the product quality, the invention refers to the standards of bulk drugs in pharmacopoeia at home and abroad, and the impurity A is taken as the known impurity to independently control the limit. The invention further improves the method for better analyzing and detecting the related substances of the calcitonin nasal spray, thereby effectively controlling the quality of the salmon calcitonin nasal spray product and improving the specificity, sensitivity and accuracy of detection.
Specifically, the invention provides an analysis method of salmon calcitonin nasal spray related substances, which comprises the following HPLC chromatographic conditions:
the mobile phase A is a mixed solution of 0.02mol/L tetramethyl ammonium hydroxide solution and acetonitrile, the volume ratio of the two is 9:1, wherein the pH of the tetramethyl ammonium hydroxide solution is adjusted to 2.5 by phosphoric acid;
the mobile phase B is a mixed solution of 0.02mol/L tetramethyl ammonium hydroxide solution and acetonitrile, the volume ratio of the two is 2:3, wherein the pH of the tetramethyl ammonium hydroxide solution is adjusted to 2.5 by phosphoric acid;
the gradient elution conditions were as follows:
time per minute Mobile phase A/%) Mobile phase B/%)
0 70-75 30-25
30 55-60 45-40
32 70-75 30-25
55 70-75 30-25
Further, the column temperature was 60-65 ℃.
The salmon calcitonin spray has more impurities, is difficult to detect, particularly has poor separation degree between main components and unknown impurities and between the impurity A and the unknown impurities, is difficult to distinguish, and the existing detection method is difficult to accurately detect related substances.
The invention unexpectedly discovers that impurities can be better separated by increasing the column temperature and adjusting the proportion of the mobile phase in 30 minutes, thereby realizing quantitative determination and remarkably improving the detection sensitivity and accuracy.
Preferably, the gradient elution conditions are as follows:
time/minute Mobile phase A/%) Mobile phase B/%)
0 72 28
30 55 45
32 72 28
55 72 28
Preferably, the column temperature is 62 ℃.
Preferably, the column used is Vydac 218TP C18,4.6 x 250mm,
Figure BDA0001913925830000051
5 μm or ZORBAX 300SB-C18, 4.6X 250mm, 5 μm.
Preferably, the flow rate of the mobile phase is 1.0 mL/min.
Preferably, the sample size is 20. mu.l.
Preferably, the detection wavelength is 220 nm.
Furthermore, the salmon calcitonin nasal spray is directly used as a test solution.
Further, a test solution diluted 90 to 110 times, preferably 100 times, with the above-mentioned mobile phase A is used as a control solution. For example, specific formulation methods include: 1.0ml of the test solution is precisely measured, placed in a 100ml measuring flask, and diluted to the scale with the mobile phase A to serve as a control solution.
Further, a system adaptability test is also included; wherein impurity A (i.e. N-acetyl-cysteinyl) is taken 1 Salmon calcitonin) as a control, adding the mobile phase A to dissolve and dilute the solution to obtain a solution containing 0.3mg of the impurity A per 1ml, and diluting the solution with the test solution 90-110 times, preferably 100 times (for example, taking 100. mu.l of the solution, adding 10ml of the test solution, and mixing well) to obtain the system suitability test solution (1).
Since there is no calcitonin C control at present, in order to better position it, the present invention first prepares salmon calcitonin control solution with the above-mentioned mobile phase a as a solvent, and then heat-treats it until it is degraded to generate impurity calcitonin C, which is then used as the system suitability test solution (2). The preferred heating conditions are heating in a water bath at 65 ℃ for 10 hours. For example, the specific method is as follows: taking appropriate amount of salmon calcitonin control, adding the above mobile phase A, dissolving, and diluting to obtain salmon calcitonin control solution with concentration of 0.2-0.5mg/ml, preferably 0.3mg/ml, heating in 65 deg.C water bath for 10 hr, cooling, and making into system applicability test solution (2).
The specific analysis method comprises the following steps: precisely measuring 20 μ l of each of the test solution and the control solution, injecting into a chromatograph, and calculating the content of related substances by peak area according to the self-control method.
20. mu.l of each of the system suitability test solution (1) and the system suitability test solution (2) was taken and injected into a chromatograph, and the chromatogram was recorded. In the chromatogram of the system applicability test solution (1), the separation degree of the impurity A peak and the main peak of the salmon calcitonin should be more than 5.0, and the tailing factor of the impurity A peak should not exceed 2.0; in the chromatogram of the system suitability test solution (2), the maximum peak between the solvent peak and the main peak is the calcitonin C peak.
Wherein the salmon calcitonin nasal spray related substance limit standard is as follows: the content of calcitonin C should not exceed 5.0%, the content of impurity A should not exceed 3.0%, the content of other single impurities should not exceed 3.0%, and the sum of all other impurities except calcitonin C should not exceed 5.0%.
The method can detect the salmon calcitonin raw material drug, crude raw material drug and salmon calcitonin preparation; the salmon calcitonin preparation comprises injection, lyophilized powder for injection and salmon calcitonin nasal spray. The method can effectively separate and quantitatively determine the existing impurities, thereby effectively controlling the quality of the salmon calcitonin nasal spray product, and has strong specificity, high sensitivity and good accuracy. The invention adopts the high performance liquid chromatography to separate and measure the salmon calcitonin and impurities in the salmon calcitonin nasal spray, and uses the ultraviolet detector to quantitatively detect the impurity content, can measure the specific numerical value of the impurity content, has high sensitivity, can directly reflect the degradation trend of a sample in the placing process, and provides higher guarantee and more exact digital basis for accurately measuring the impurity content and effectively controlling the product quality.
Drawings
FIG. 1: example 1 typical chromatogram of a test solution.
FIG. 2: example 1 system applicability a typical chromatogram of a test solution (1).
FIG. 3: experimental example chromatographic conditions 1 typical chromatogram.
FIG. 4: experimental example chromatographic conditions 2 typical chromatograms.
FIG. 5 is a schematic view of: experimental example chromatographic conditions 3 typical chromatogram.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Unless otherwise specified, the following instruments and reagents were used:
a chromatograph: agilent 1260 liquid chromatograph.
A detector: a DAD detector for detecting a wavelength of 220 nm;
mobile phase A: 0.022mol/L tetramethylammonium hydroxide solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile
(9:1, v/v); and (3) mobile phase B: 0.02mol/L tetramethylammonium hydroxide solution (pH adjusted with phosphoric acid)
Is 2.5) -acetonitrile (2:3, v/v)
And (3) chromatographic column: vydac 218TP C18, 4.6X 250mm,
Figure BDA0001913925830000071
5μm。
a chromatographic column: ZORBAX 300SB-C18, 4.6X 250mm, 5 μm.
The specific method can refer to high performance liquid chromatography (addendum VD of 2010 version in Chinese pharmacopoeia).
The test solutions used below were salmon calcitonin nasal spray, supplied by silver grain pharmaceutical llc, under the accession numbers 1207011, 1207021, 1207031, 1408021, 1408031, 1408041.
1.0ml of the test solution (salmon calcitonin nasal spray) is precisely measured, placed in a 100ml measuring flask, and diluted to the scale with the mobile phase A to serve as a control solution.
Salmon calcitonin control, supplied by the chinese food and drug assay institute, lot No.: 140665-201103.
Impurity A (N-acetyl-cysteinyl) 1 Salmon calcitonin) control, USP control, lot No.: FOK 102.
Taking impurity A (N-acetyl-cysteinyl) 1 Salmon calcitonin) as a control, adding mobile phase A to dissolve and dilute the solution to obtain a solution containing 0.3mg per 1ml, adding 10ml of the test solution into 100. mu.l of the solution, and mixing to obtain the system suitability test solution (1).
Taking a proper amount of salmon calcitonin control, adding the mobile phase A to dissolve and dilute the salmon calcitonin control to prepare a solution containing about 0.3mg of salmon calcitonin per 1ml, heating the solution in a water bath at 65 ℃ for 10 hours, and cooling the solution to obtain a system applicability test solution (2).
Example 1 high Performance liquid chromatography assay of salmon calcitonin nasal spray-related substance
HPLC chromatographic conditions: the column temperature is 62 ℃; a DAD detector with a detection wavelength of 220 nm; a chromatographic column: ZORBAX 300SB-C18, 4.6X 250mm, 5 μm.
Gradient elution procedure:
time/minute Mobile phase A/%) Mobile phase B/%)
0 72 28
30 55 45
32 72 28
55 72 28
Precisely measuring 20 μ l of each of the test solution and the control solution, injecting into a chromatograph, recording chromatogram as shown in figure 1, and calculating related substance content by peak area according to self control method. Wherein, the content of calcitonin C is 1.06%, the content of impurity A is 0.15%, the content of other single impurities is not more than 3.0%, and the sum of all other impurities except calcitonin C is not more than 5.0%.
Test of system suitability
20. mu.l of each of the system suitability test solution (1) and the system suitability test solution (2) was taken and injected into a chromatograph, and the chromatogram was recorded. In the chromatogram 2 of the system suitability test solution (1), the separation degree of the impurity a peak from the main peak of salmon calcitonin is more than 5.0, and the tailing factor of the impurity a peak is not more than 2.0; the maximum peak between the solvent peak and the main peak is the calcitonin C peak. The specific results are shown in the following table:
Figure BDA0001913925830000081
and (4) conclusion: under the chromatographic conditions, the calcitonin C, the impurity A and the main peak are well separated, and the test requirement of the applicability of the system is met.
Examples of the experiments
By consulting USP, BP, EP and CP, the chromatographic conditions adopted in pharmacopoeia standards of various countries are basically the same except that the gradient elution time is slightly different. There are two main types of gradient elution time, one is used for the examination of relevant substances in raw materials, the gradient elution time is 55 minutes, and the other is used for the examination of relevant substances in preparations, the elution time is 30 minutes. In order to understand the difference between the two chromatographic conditions, the invention adopts different chromatographic columns to investigate the two chromatographic conditions, and the results are as follows:
A. chromatographic conditions 1:
chromatograph: agilent 1260 liquid chromatograph
A detector: DAD detector with detection wavelength of 220nm
Mobile phase A: 0.022mol/L tetramethylammonium hydroxide solution (pH 2.5 adjusted with phosphoric acid) -acetonitrile (9:1)
And (3) mobile phase B: 0.02mol/L tetramethylammonium hydroxide solution (pH 2.5 adjusted with phosphoric acid) -acetonitrile (2:3)
And (3) chromatographic column: vydac 218TP C18, 4.6X 250mm,
Figure BDA0001913925830000091
5μm
column temperature: 40 deg.C
Gradient program:
time/minute Mobile phase A/%) Mobile phase B/%)
0 65 35
21 43 57
21.01 65 35
30 65 35
A typical chromatogram is shown in FIG. 3.
B. Chromatographic conditions 2:
the only difference from chromatographic condition 1 is: the column temperature was 62 ℃ and gradient elution was performed using the following conditions:
time per minute Mobile phase A/%) Mobile phase B/%)
0 72 28
30 48 52
32 72 28
55 72 28
A typical chromatogram is shown in FIG. 4.
C. Chromatographic conditions 3
The only difference from chromatographic condition 1 is: the column temperature was 62 ℃ and the column ZORBAX 300SB-C18 was used,
4.6X 250mm, 5 μm, gradient elution was performed under the following conditions:
time (minutes) A％ B％
0 72 28
30 48 52
32 72 28
55 72 28
A typical chromatogram is shown in FIG. 5.
As can be seen from fig. 3-5, there was no substantial difference in the separation capacity between the two different columns, as compared to the slightly better separation capacity of ZORBAX 300SB-C18, and a subsequent study was performed using this type of column. The number of theoretical plates of calcitonin C in FIG. 3 is 4807, the number of theoretical plates of calcitonin C in FIG. 4 is 16488, and the number of theoretical plates of calcitonin C in FIG. 5 is 21225; in addition, chromatographic condition 1 initial gradient change was (65-43)/21; chromatographic condition 2 the initial gradient varied (72-48)/30, with essentially similar variations, so it can be seen that column temperature had a significant effect on theoretical plate number. From the results obtained by the two column temperatures, the theoretical plate number of calcitonin C can be well improved by the higher column temperature, so that subsequent research is carried out by adopting the high column temperature.
From the results obtained by the two gradient elution modes, the main peak retention time is a little longer for the related substance inspection chromatographic conditions of the bulk drug, but the separation condition of each impurity is far better than the gradient condition used for the preparation, and the two chromatographic conditions can separate the salmon calcitonin C well, but the two conditions have larger difference for detecting other related peptides, so the subsequent research is carried out by adopting the condition with longer elution time in the research.
It has surprisingly been found that adjusting the proportion of mobile phase at 30 minutes enables a better separation of impurities. The specific chromatographic conditions were as follows:
chromatograph: agilent 1260 liquid chromatograph
A detector: DAD detector with detection wavelength of 220nm
Mobile phase A: 0.02mol/L tetramethylammonium hydroxide solution (pH 2.5 adjusted with phosphoric acid) -acetonitrile (9:1)
Mobile phase B: 0.02mol/L tetramethylammonium hydroxide solution (pH 2.5 adjusted with phosphoric acid) -acetonitrile (2:3)
A chromatographic column: ZORBAX 300SB-C18, 4.6X 250mm, 5 μm
Column temperature: 62 deg.C
Gradient elution procedure:
time/minute Mobile phase A/%) Mobile phase B/%)
0 72 28
30 55 45
32 72 28
55 72 28
A typical chromatogram of the obtained test solution is shown in FIG. 1.
According to the detection results, the method provided by the invention can be used for effectively analyzing and quantitatively determining the impurities possibly existing in the salmon calcitonin nasal spray, so that the product quality is effectively controlled, and the method is strong in specificity, high in sensitivity and good in accuracy.
Description of Standard Limit settings
The european pharmacopoeia and the us pharmacopoeia salmon calcitonin standard list 4 known impurities, respectively A, B, C, D, and one known impurity salmon calcitonin C was also introduced in the standard text. For information on the specific structure of each impurity, see above.
Referring to domestic and foreign pharmacopoeias salmon calcitonin raw material and injection, salmon calcitonin nasal spray domestic new drug conversion standard and related substance limit requirements of the import registration standard of the same products, the impurity limit is set to be 5.0 percent of calcitonin C, 3.0 percent of impurity A, 3.0 percent of other single impurities and 5.0 percent of impurities, and the setting of each limit is described as follows:
calcitonin C: the impurities are main degradation products of the product, are easy to generate in solution and are not easy to appear in raw materials, and the determination result shows that the impurities are not detected after the raw materials are placed for two years, so that the limit of the impurities is not independently controlled in the raw materials in the existing domestic and foreign standards, but the limit is controlled to be 7.0% in the injection. The imported product of salmon calcitonin nasal spray only controls the impurities, the limit is 5.0 percent, and the limit is not separately specified in the national nasal spray conversion standard. According to the limit and the actual measurement result of the sample, the calcitonin C limit is not more than 5.0 percent.
Impurity A: the only synthetic impurity detected in the four known impurities (the impurities A, B, C and D) has no independent limit of the impurity A in the salmon calcitonin nasal spray product at home and abroad. In order to strictly control the product quality, the standard is drawn up and referred to the standards of bulk drugs in pharmacopoeia at home and abroad, and the impurity is used as a known impurity to independently control the limit, wherein the limit is specified to be not more than 3.0%.
Other individual impurities: the limit is not more than 3.0 percent which is consistent with the standard regulation of each raw material and injection.
The remaining total impurities: the product contains other degradation products and synthetic impurities besides calcitonin C, and the limit of the other total impurities is set to be less than 5.0 percent by referring to the domestic and foreign pharmacopoeia standards of salmon calcitonin injection.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims (9)

1. An analysis method of salmon calcitonin nasal spray related substances adopts high performance liquid chromatography for detection, and is characterized in that the salmon calcitonin nasal spray is directly used as a sample solution to be detected for detection;
the HPLC chromatographic conditions used were as follows:
the column used was Vydac 218TP C18,4.6 x 250mm,
Figure FDF0000017444370000011
5 μm or ZORBAX 300SB-C18, 4.6 × 250mm, 5 μm;
the mobile phase A is a mixed solution of 0.02mol/L tetramethyl ammonium hydroxide solution and acetonitrile, the volume ratio of the two is 9:1, wherein the pH of the tetramethyl ammonium hydroxide solution is adjusted to 2.5 by phosphoric acid;
the mobile phase B is a mixed solution of 0.02mol/L tetramethylammonium hydroxide solution and acetonitrile, the volume ratio of the two is 2:3, wherein the pH of the tetramethylammonium hydroxide solution is adjusted to 2.5 by phosphoric acid;
the gradient elution conditions were as follows:
time/minute Mobile phase A/%) Mobile phase B/%) 0 72 28 30 55 45 32 72 28 55 72 28
；
The column temperature is 60-65 ℃; the flow rate of the mobile phase is 0.8-1.2 mL/min; the sample injection amount is 5-30 mul; the detection wavelength is 210-254 nm;
the related substances comprise impurities A and calcitonin C:
Figure FDF0000017444370000012
impurity A: r 1 ＝CO-CH 3 ，R 2 ＝NH 2 X-Leu; acetyl salmon calcitonin;
Figure FDF0000017444370000013
calcitonin C;
the related substances also include the following impurities:
Figure FDF0000017444370000021
impurity B: r is 1 ＝H，R 2 ＝NH 2 X-Leu; [ 9-D-leucine]-salmon calcitonin;
Figure FDF0000017444370000022
impurity C: des-22-tyrosine salmon calcitonin
Figure FDF0000017444370000023
Impurity D: oxyacetyl salmon calcitonin.
2. The analytical method according to claim 1, wherein the column temperature is 62 ℃.
3. The method according to claim 1, wherein the salmon calcitonin nasal spray is used as a test solution; the test solution diluted 90-110 times with the mobile phase A is used as a control solution.
4. The method according to claim 1, wherein the salmon calcitonin nasal spray is used as a test solution; the test solution diluted 100 times with the mobile phase a was used as a control solution.
5. The assay of claim 1, further comprising a system suitability test; wherein N-acetyl-cysteinyl is taken 1 -a proper amount of salmon calcitonin control substance is dissolved and diluted by adding the mobile phase A to prepare a solution of 0.3mg/ml, and then the solution is diluted by 90-110 times by using the test solution to be used as a system suitability test solution (1);
taking a proper amount of salmon calcitonin reference substance, adding the mobile phase A to dissolve and dilute to prepare salmon calcitonin reference substance solution with the concentration of 0.2-0.5mg/ml, heating to degrade to generate impurity calcitonin C, and cooling to obtain the system applicability test solution (2).
6. The assay of claim 5, wherein the systemic suitability test is performed using N-acetyl-cysteinyl 1 A proper amount of salmon calcitonin reference substance is dissolved and diluted by adding the mobile phase A to prepare a solution of 0.3mg/ml, and then the solution is diluted by 100 times by using the test substance solution to serve as a system applicability test solution (1);
taking a proper amount of salmon calcitonin reference substance, adding the mobile phase A to dissolve and dilute to prepare salmon calcitonin reference substance solution with the concentration of 0.3mg/ml, heating to degrade to generate impurity calcitonin C, and cooling to obtain the system applicability test solution (2).
7. The assay of claim 1, wherein the flow rate of the mobile phase is 0.9-1.1 mL/min; and/or the sample amount is 10-30 mul; and/or the detection wavelength is 220 nm.
8. The assay of claim 7, wherein the flow rate of the mobile phase is 1.0 mL/min; and/or the sample amount is 20 mul.
9. The analysis method according to claim 1, comprising precisely measuring 20. mu.l each of the test solution and the control solution, injecting into a chromatograph, recording the chromatogram, and calculating the content of the substance in terms of peak area according to a self-control method.
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