CN107907603B

CN107907603B - Method for measuring tryptophan related substances in amino acid injection

Info

Publication number: CN107907603B
Application number: CN201711050017.8A
Authority: CN
Inventors: 尹冬丽; 韩瑶
Original assignee: Huaren Pharmaceutical Co Ltd
Current assignee: HUBEI HUAREN TONGJI PHARMACEUTICAL CO.,LTD.
Priority date: 2017-10-31
Filing date: 2017-10-31
Publication date: 2021-01-05
Anticipated expiration: 2037-10-31
Also published as: CN107907603A

Abstract

The invention provides a method for measuring amino acid injection tryptophan related substances, and relates to the technical field of medicines; the determination method is used for determining the content of tryptophan related substances in the amino acid injection, adopts high performance liquid chromatography, adopts a chromatographic column with octadecylsilane chemically bonded silica as a stationary phase, and adopts three mobile phases: the mobile phase A is 0.01mol/L sodium dihydrogen phosphate, and the PH value is adjusted to 7.2 by sodium hydroxide solution; the mobile phase B is obtained by mixing acetonitrile, methanol and water according to the volume ratio of 45:45: 10; the mobile phase C is obtained by mixing methanol and water according to the volume ratio of 10:90, gradient elution is carried out, analysis and calculation are carried out in a mode of tryptophan relative retention time, and tryptophan related substances in the amino acid injection are separated and determined.

Description

Method for measuring tryptophan related substances in amino acid injection

Technical Field

The invention provides a method for measuring amino acid injection tryptophan related substances, and relates to the technical field of medicines.

Background

The amino acid transfusion has various varieties, different varieties and quantities of amino acid, electrolyte and the like contained in the varieties, different applications and wide clinical application. Tryptophan is one of essential amino acids, can be converted into an important neurotransmission substance in human brains, namely 5-hydroxytryptamine, is one of main components in amino acid injection, and is sensitive to light, heat, oxygen and the like, so that in the preparation process of the amino acid injection, if links such as an oxygen control process, a sterilization process and the like are not reasonably controlled, the tryptophan is easily degraded to generate related substances, and the tryptophan degradation rate, namely the proportion of the tryptophan related substances in the amino acid injection can reflect the quality of amino acid injection products. In EP and USP pharmacopoeia, related substances in tryptophan raw material medicines are introduced, but no literature report of tryptophan related substances is found in amino acid injection.

Disclosure of Invention

The invention solves the technical problem that the prior art lacks a method for determining tryptophan related substances in amino acid injection, and provides a method for determining tryptophan related substances in amino acid injection with high efficiency, accuracy and cost, which comprises the following steps: performing gradient elution by high performance liquid chromatography with chromatographic column using octadecylsilane chemically bonded silica as stationary phase under the following mobile phase conditions; the method comprises three mobile phases, wherein the mobile phase A is 0.01mol/L sodium dihydrogen phosphate, and the PH value is adjusted to 7.0-7.4 by using a sodium hydroxide solution; the mobile phase B is obtained by mixing acetonitrile, methanol and water according to the volume ratio of 45:45: 10; the mobile phase C is obtained by mixing methanol and water according to the volume ratio of 10: 90; the gradient of the mobile phase was set as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)	Mobile phase C (%)	Flow rate (ml/min)
					0	100	0	0	1.2
30	45	55	0	1.2
					35	0	100	0	1.2
35.5	0	0	100	1.2
					36	0	0	100	1.5
40	0	0	100	1.5
					42.5	100	0	0	1.5
45	100	0	0	1.2
					48	100	0	0	1.2

More preferably, the column temperature is 40 ℃ and the detection wavelength is 220 nm.

As a further preference, the PH of the mobile phase a is 7.2.

The invention establishes an analysis and determination method for tryptophan related substances in amino acid injection by referring to a detection method for tryptophan related substances in raw material medicines in EP, BP and USP pharmacopoeia. In the analysis process, acetyl tryptophan is used as a self control, and the analysis calculation is carried out in a mode of relative retention time of tryptophan, so that the sum of the peak areas of specified impurities is not more than 5 times (1.0%) of the peak area of an acetyl tryptophan control product. The results are verified by methodology, tryptophan related substances in amino acid injection (compound amino acid injection (18AA), compound amino acid injection (18AA-II), compound amino acid injection (17AA), compound amino acid (15) dipeptide (2) injection and the like) can be effectively determined, oxygen control process, sterilization process and the like in the preparation process are monitored, and the quality of finished products is reflected.

Drawings

FIG. 1 methionine chromatogram;

FIG. 2 (hydrochloric acid) histidine chromatogram;

FIG. 3 aspartic acid chromatogram;

FIG. 4 alanine chromatogram;

FIG. 5 valine chromatogram;

FIG. 6 (hydrochloric acid) lysine chromatogram;

FIG. 7 leucine chromatogram;

FIG. 8 threonine chromatogram;

FIG. 9 tyrosine chromatogram;

FIG. 10 (arginine hydrochloride) chromatogram;

FIG. 11 isoleucine chromatogram;

FIG. 12 proline chromatogram;

FIG. 13 phenylalanine chromatogram;

FIG. 14 Glycine chromatogram;

FIG. 15 serine chromatogram;

FIG. 16 glutamic acid chromatogram;

FIG. 17 tryptophan chromatograms;

FIG. 18 chromatogram of Tryptophan related substance of Compound amino acid injection (18 AA);

FIG. 19 chromatogram of tryptophan related substances in Compound amino acid injection (18 AA-2);

FIG. 20 chromatogram of tryptophan related substances in compound amino acid (15) dipeptide (2) injection;

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following examples are provided to further illustrate the method for detecting tryptophan related substances in amino acid injection according to the present invention, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples, and all the technologies realized based on the above contents of the present invention are within the scope of the present invention.

Example 1

Directly sampling the product; an appropriate amount of tryptophan control was also precisely weighed and made into a solution containing about 0.9mg of tryptophan per 1ml with water. According to the determination of high performance liquid chromatography, octadecylsilane chemically bonded silica is used as a filler, a chromatographic column of a stationary phase is used, acetyltryptophan is used as a self control to detect related substances of the tryptophan, the number of mobile phases is three, the mobile phase A is 0.01mol/L sodium dihydrogen phosphate, the pH value is adjusted to 7.2 by using a sodium hydroxide solution, and the number of mobile phases B is acetonitrile, methanol and water according to the ratio of 45:45:10, and the mobile phase C is methanol and water according to the volume ratio of 10:90, the column temperature is 40 ℃, and the detection wavelength is 220 nm; gradient elution was performed as follows.

Precisely measuring the sample solution and tryptophan reference solution by 20 μ l each, injecting into a liquid chromatograph, and recording chromatogram; an appropriate amount of the acetyl tryptophan control substance was weighed, dissolved in water and diluted to a solution containing about 2.0. mu.g of acetyl tryptophan per 1ml as a control solution, and the measurement was performed in the same manner. The peaks in the chromatogram before 0.54 times the relative retention time of tryptophan are not counted; after the time, except the tryptophan peak and the peak of tryptophan which are 0.78 times of the relative retention time, if an impurity peak exists before 30min, the sum of the peak areas of the impurity peaks is not more than 5 times (1.0%) of the peak area of the acetyl tryptophan control product. Any peak in the test solution that is less than 0.1 times the area of the major peak of the acetyltryptophan control solution is negligible.

In the examples, the calculation formula of the tryptophan related substances, namely impurities, in the amino acid injection is as follows:

r_U: total area of tryptophan impurities in the test solution; r is_S: acetyl tryptophan peak area; c. C_S: acetyl tryptophan concentration (mg/ml); c. C_U: the prescribed concentration of tryptophan.

The following items were examined for the measurement method of tryptophan related substances in amino acid injection:

1. confirmation of relative Retention time of Tryptophan

The prescription amount of 18 amino acid solutions is prepared, the sample injection is carried out according to the liquid phase conditions described in the embodiment, the chromatogram is shown in the attached figures 1-17, and the statistical content is shown in tables 1 and 2.

TABLE 118 amino acid retention times

Item	Retention time
		Cystine	Does not peak
(hydrochloric acid) lysine	2.108
		Aspartic acid	2.115
Glutamic acid	2.141
		Serine	2.193
Glycine	2.182
		Alanine	2.236
Threonine	2.300
		(hydrochloric acid) arginine	2.329
(hydrochloric acid) histidine	2.649
		Proline	2.682
Valine	3.252
		Methionine	4.322
Isoleucine	5.582
		Tyrosine	5.655
Leucine	5.833
		Phenylalanine	8.851
Tryptophan	11.328

TABLE 2 relative Retention time schedules of Tryptophan to methionine etc

Contrast item	Relative retention time of tryptophan
		Isoleucine	0.493
Tyrosine	0.499
		Leucine	0.515
Phenylalanine	0.781

As can be seen from the contents of table 1 and table 2, the peak before 0.54 times the relative retention time of tryptophan and the peak 0.78 times the relative retention time of tryptophan (phenylalanine) in the chromatogram were all negative sample peaks, and no tryptophan related substance was contained. The peak after 0.54 times of the relative retention time of tryptophan and the peak after 0.78 times of the relative retention time of tryptophan were removed to obtain tryptophan related substance, and the total area of tryptophan related substance, i.e. impurities, was calculated and used

Tryptophan impurity% is obtained by a formula, so that the quality of the test sample is reflected.

2. Selection of self-control

According to the detection method of tryptophan raw material drug related substances in USP, BP and EP, acetyl tryptophan is used as a self control to detect tryptophan related substances.

Under the liquid phase conditions of example 1, the area of the tryptophan peak at 2. mu.g/ml is 307469, and the area of the acetyl tryptophan peak at 2. mu.g/ml is 241138; from the above experimental data, it is possible to select acetyltryptophan as the self-control in this example to be more stringent.

3. Methodology study

(1) Chromatographic conditions and System suitability test

System applicability solution preparation: an appropriate amount of acetyltryptophan control was taken to prepare a solution containing about 2.0. mu.g of acetyltryptophan per 1ml as a system suitability solution.

The operation method comprises the following steps: and taking the system applicability solution, injecting 20 mu l of the solution for detection, repeating the detection for six times, and recording a chromatogram map. RSD calculated for the peak-to-peak area of acetyltryptophan was 0.5%; RSD of main peak retention time was 0.01%.

(2) Specificity (interference test)

According to the detection method under related substance items, 20 μ l of each of the aqueous solution, mobile phase solution, acetyltryptophan reference solution, tryptophan reference solution, negative solution (prepared according to the prescription without tryptophan) and preparation sample is injected into a liquid chromatograph, and chromatogram is recorded.

The results prove that the water solution and the mobile phase solution have no interference on the impurity research, while the negative solution has interference and needs blank deduction.

(3) Detection limit

The acetyl tryptophan control solution was diluted to different concentrations and assayed based on a 3:1 signal to noise ratio, at an acetyl tryptophan concentration of 0.51. mu.g/ml.

(4) Limit of quantification

The acetyl tryptophan control solution was diluted to different concentrations and assayed based on a 10:1 signal to noise ratio, at an acetyl tryptophan concentration of 1.82. mu.g/ml. 6 test solutions with the lowest quantitative limit concentration are prepared and put into 1 needle respectively, the RSD of the acetyl tryptophan peak retention time is 0.01 percent, and the RSD of the peak area is 0.5 percent.

(5) Accuracy test

Respectively and precisely weighing 10mg, 10mg and 10mg of acetyl tryptophan reference substances; 20mg, 20 mg; 30mg, 30 mg; placing in 9 100ml measuring bottles, adding negative solution (prepared according to the prescription and not containing tryptophan) for dissolving, fixing the volume, and shaking up. And precisely measuring 1.00ml of the solution respectively, placing the solution into 9 100ml measuring bottles, adding a negative solution (which is prepared according to a prescription and does not contain tryptophan) for dissolving, fixing the volume, and shaking up to obtain a solution to be measured for accuracy. According to the detection method under the related substance item, the chromatogram is recorded by the method, the recovery rate and the relative standard deviation of the recovery rate are calculated, and the average recovery rate under each concentration is between 80 and 120 percent. The results are shown in Table 3:

TABLE 3 accuracy test results for acetyltryptophan

The results show that: the average recovery rate of the method for measuring the content of acetyltryptophan is 108.1 percent, the RSD is 3.8 percent, and the accuracy is good in the range of 50 to 150 percent of the marked amount.

(6) Precision test-repeatability test, intermediate precision test

And (3) repeatability test: taking B1401001 batches of compound amino acid injection (18AA), filling the stock solution into 6 liquid phase bottles for sample injection according to a detection method under related substance items, recording a chromatogram, and calculating the relative standard deviation, wherein the RSD is less than or equal to 15%. Loading the stock solution into 6 liquid bottles, sampling, measuring by different personnel on different instruments according to the method, recording chromatogram, and calculating relative standard deviation of measured content. Compared with the results of repeated experiments, the RSD of the obtained 12 data is less than or equal to 20%. The results are reported in table 4 below:

TABLE 4 results of precision test of related substances of tryptophan

As can be seen from Table 4: in the repeatability test, the RSD of the tryptophan related substance determination of 6 samples is 12 percent, which indicates that the repeatability of the content determination method is good. In the intermediate precision test, the RSD of 12 samples of tryptophan related substances is 12 percent, which shows that the intermediate precision of the content measuring method is good.

(7) Durability

Comparison of different column temperatures

Taking the compound amino acid injection (18AA) of the B1401001 batch, injecting samples at the column temperatures of 36 ℃, 39 ℃ and 42 ℃ respectively and recording chromatograms according to the detection method under the relevant substance items and other chromatographic conditions. The test results are shown in Table 5:

TABLE 5 test results of the effect of different column temperatures on tryptophan related substances

The results show that: under different detection temperatures of the condition, the compound amino acid injection (18AA) has the advantages that the absolute value of tryptophan related substances is within +/-0.1 percent, and the stability result is good.

② comparison of pH of different mobile phases

Sampling B1401001 batch of compound amino acid injection (18AA) samples, keeping other chromatographic conditions unchanged, setting the pH values of the mobile phase A to be 7.0, 7.2 and 7.4 respectively, and recording chromatograms. The test results are shown in Table 6:

TABLE 6 test results of the effect of different mobile phase pH values on tryptophan related substances

The results show that: under different mobile phase pH values of the compound amino acid injection (18AA), the absolute value of tryptophan related substances is within +/-0.1%, and the stability result is good.

Comparison of different detection wavelengths

Sampling B1401001 batches of compound amino acid injection (18AA) samples, keeping other chromatographic conditions unchanged, setting detection wavelengths of 215nm, 220nm and 225nm respectively, injecting samples, and recording chromatograms. The stability test results are shown in table 7:

TABLE 7 test results of the influence of different detection wavelengths on tryptophan related substances

The results show that: under different detection wavelengths of the condition, the compound amino acid injection (18AA) has the advantages that the absolute value of tryptophan related substances is within +/-0.1%, and the stability result is good.

Comparison of different flow rates

Sampling B1401001 batches of compound amino acid injection (18AA) samples under the same other chromatographic conditions, respectively setting the flow rates of the samples to be 1.0ml/min, 1.2ml/min and 1.4ml/min, and recording the chromatogram. The test results are shown in Table 8:

table 8 test results for examining the influence of different flow rates on tryptophan related substances

The results show that: under different flow rates of the compound amino acid injection (18AA) under the condition, the absolute value of tryptophan related substances is within +/-0.1%, and the stability result is good.

(8) Stability test of solution

Taking the compound amino acid injection (18AA) of the B1401001 batch, respectively measuring once in 0 hour, 2 hours, 4 hours, 6 hours and 8 hours according to the detection method under the relevant substance item, calculating the relative standard deviation of the impurity peak area, and obtaining the result shown in the following table 9.

TABLE 9 stability test results of Tryptophan related substances in Compound amino acid injection (18AA)

The results show that: the tryptophan related substances in the compound amino acid injection (18AA) are determined within 8h, the absolute value of the tryptophan related substances is within +/-0.1%, and the solution stability result is good.

(9) The results of the methodological validation are shown in table 10 below.

TABLE 10 summary of methodological validation results

4. Sample assay

(1) The tryptophan related substance content of the compound amino acid injection (18AA) reference preparation and the 3 batches of compound amino acid injection (18AA) samples in the test production were determined according to the tryptophan related substance determination method, and the results are shown in Table 11.

TABLE 11 measurement results of Tryptophan related substance content of Compound amino acid injection (18AA)

The results show that: the peak area of the tryptophan related substance of the reference preparation and the 3 batches of samples is not larger than 5 times (1.0%) of the peak area of the acetyl tryptophan control product, and the result is good.

(2) The results of the compound amino acid injection (18AA), the compound amino acid injection (18AA-II) and the compound amino acid (15) dipeptide (2) injection with 24-month stability were determined according to the tryptophan related substance determination method, and are shown in table 12 below, wherein the chromatograms of the compound amino acid injection (18AA), the compound amino acid injection (18AA-II) and the compound amino acid (15) dipeptide (2) injection are respectively shown in fig. 18, fig. 19 and fig. 20.

TABLE 12 measurement results of tryptophan related substances in different amino acid injections

The results show that: the compound amino acid injection (18AA), the compound amino acid injection (18AA-II), the compound amino acid injection (17AA) with the concentration of 15 percent and the compound amino acid (15) dipeptide (2) injection have good results, and the peak areas of tryptophan related substances are not more than 5 times (1.0 percent) of the peak area of an acetyl tryptophan reference substance.

While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods and applications described herein, as well as other variations and combinations of the techniques described herein, may be made and used without departing from the spirit and scope of the invention. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention.

Claims

1. A method for determining amino acid injection tryptophan related substances is characterized in that high performance liquid chromatography is adopted, acetyl tryptophan is selected as self contrast, a chromatographic column with octadecylsilane chemically bonded silica as a stationary phase is used, and gradient elution is carried out according to the following mobile phase conditions; the method comprises three mobile phases, wherein the mobile phase A is 0.0lmol/L sodium dihydrogen phosphate, and the PH value is adjusted to 7.2 by using sodium hydroxide solution; the mobile phase B is obtained by mixing acetonitrile, methanol and water according to the volume ratio of 45: 10; the mobile phase C is obtained by mixing methanol and water according to the volume ratio of 10:90, the column temperature is 40 ℃, the detection wavelength is 220nm, and the gradient of the mobile phase is set as follows:

。

2. The method of claim 1, wherein the peak after 0.54 time of the tryptophan relative retention time is a tryptophan-related substance peak excluding the peak 0.78 time of the tryptophan relative retention time and the tryptophan peak.