WO2023123309A1 - Method for measuring amino acid impurities in special solvent for butanedisulfonic acid adenosine methionine for injection - Google Patents

Abstract

A method for measuring amino acid impurities in a special solvent for butanedisulfonic acid adenosine methionine for injection. The measurement method comprises: preparing a standard solution and a sensitivity solution of each amino acid impurity (DL-aminocaprolactam, phenylalanine, leucine, and threonine); preparing a sample to be measured: diluting a special solvent for butanedisulfonic acid adenosine methionine for injection with diluents into a solution having a certain concentration to form said sample, the diluents being methanol and purified water at a volume ratio of 60 : 40; preparing a mobile phase into a mobile phase A (5:95) and a mobile phase B (50:50) by using a buffer salt (the pH of 10mM ammonium formate being adjusted to 3.0 with formate acid) and acetonitrile according to a certain ratio; and carrying out sample measurement by using a high-performance liquid chromatography-mass spectrometer, so that the content of each amino acid impurity in the solvent special for the butanedisulfonic acid adenosine methionine for injection can be measured by the method.

Description

A method for detecting amino acid impurities in adenosylmethionine succinate injection special solvent technical field

The invention relates to the technical field of quality control of a special solvent for ademetionine succinate for injection, in particular to a method for detecting amino acid impurities in a special solvent for ademetionine succinate for injection.

Background technique

Adenosylmethionine is a physiologically active molecule present in all tissues and fluids of the human body. As a methyl donor (transmethylation) and a precursor of physiological sulfur compounds (such as cysteine, taurine, glutathione and coenzyme A, etc.) biochemical reactions. Freeze-dried powder for injection (adenosylmethionine succinate for injection) must be dissolved with the attached solvent before use, a special solvent for adenosylmethionine succinate for injection.

The special solvent for adenosylmethionine succinate for injection is a sterilized solution of L-lysine, and the content of L-lysine (C ₆ H ₁₄ N ₂ O ₂ ) in each tube (5ml) should be 385~450mg. The chemical structure of L-lysine is as follows:

Lysine is the basic unit of protein. It is the raw material for the synthesis of human hormones, enzymes and antibodies. It participates in human metabolism and various physiological activities. Lysine is an essential amino acid for the human body and is basically found in various amino acid infusion formulas.

Most amino acids are obtained through fermentation process, and most of the impurities produced are other amino acids. According to the technological process of L-lysine, other amino acid impurities that may exist in L-lysine include phenylalanine, leucine, threonine and DL-aminocaprolactam hydrochloride, etc. In the existing detection technology, only L-arginine in the special solvent for adenosylmethionine succinate for injection is detected. It uses thin-layer chromatography for limit detection rather than quantitative detection. After the sample solution climbs the plate of thin-layer chromatography and develops color with a chromogenic agent, it is visually detected, and the sensitivity is low. Moreover, the TLC method cannot detect the impurities of phenylalanine, leucine, threonine and DL-aminocaprolactam hydrochloride that may exist in the special solvent for adenosylmethionine succinate for injection. Thin-layer chromatography needs to use more organic toxic developer and chromogenic reagent, and the cumbersome operation steps can introduce more experimental systematic errors, and it is difficult to achieve quantitative detection at lower concentrations. Therefore, it is necessary to develop a simple and reliable detection method to improve the existing impurity detection method. Compared with the existing method for detecting impurities in adenosylmethionine succinate methionine special solvent for injection, the present invention changes the tedious detection steps of thin-layer chromatography detection, avoids the use of organic toxic reagents, and is a great benefit for researchers. A more wholesome approach, while allowing effective quality control of the amount of multiple amino acid impurities.

Contents of the invention

The main purpose of the present invention is to provide a method for detecting amino acid impurities in the special solvent for ademetionine succinate for injection, so as to solve the problem of amino acid impurities in the special solvent for adenosylmethionine succinate for injection in the prior art Problems that cannot be effectively quantified.

In order to achieve the above object, according to one aspect of the present invention, a method for detecting amino acid impurities in ademethionine succinate injection special solvent is provided, which is characterized in that it includes:

Step S1: Dissolving the standard substances of DL-aminocaprolactam, phenylalanine, leucine, and threonine in a diluent to obtain a standard solution;

Step S2: dissolving the special solvent for adenosylmethionine succinate for injection with a diluent to obtain a sample solution;

Step S3: Using high performance liquid chromatography-mass spectrometry to detect the standard solution obtained in step S1 and the sample solution obtained in step S2.

Furthermore, a method for detecting amino acid impurities in a special solvent for adenosylmethionine succinate for injection of the present invention, the amino acid impurities include: phenylalanine, leucine, DL-aminocaprolactam, threonine The detection method includes: preparing standard solutions of various amino acid impurities; preparing sensitivity solutions; preparing samples to be tested, taking a special solvent for adenosylmethionine succinate for injection, diluting with a diluent to a certain concentration, and then mixing to obtain a sample solution; For sample detection, the standard solution and the sample solution to be detected are detected by high performance liquid chromatography-mass spectrometry to obtain the characteristic ion peak area of each amino acid impurity; and the characteristic ion peak area and standard product detected according to the sample detection The measured characteristic ion peak area is converted to obtain the impurities of each amino acid in the special solvent for adenosylmethionine succinate for injection.

Further, the above-mentioned process of preparing the standard solution includes: weighing 5 mg each of phenylalanine, leucine, and threonine, and 6.4 mg of DL-aminocaprolactam hydrochloride (equivalent to 5 mg of DL-aminocaprolactam) In a 25ml volumetric flask, dissolve with diluent and make the volume up to the mark, and mix well to obtain stock solution A. Precisely pipette 0.4ml of stock solution A into a 10ml volumetric flask, dilute to the mark with diluent, and mix well to obtain stock solution B. Precisely pipette 0.5ml of stock solution B into a 10ml volumetric flask, dilute to the mark with a diluent, and mix well to obtain a standard with a concentration of 0.4μg/ml for phenylalanine, leucine, threonine, and DL-aminocaprolactam product solution.

Further, the process of the above-mentioned sensitivity solution includes: pipetting 3.0ml of the above-mentioned standard solution, adding a diluent to dilute to 10ml, and mixing evenly. Further, the above-mentioned diluent is a mixed solution of methanol and water mixed at a volume ratio of 60:40.

Further, the above-mentioned preparation of the sample to be tested includes: taking 1 bottle of adenosylmethionine dimethylsulfonate special solvent for injection (specification 5ml: 428mg), pipetting 0.4ml and diluting it to 10ml with a diluent, and mixing to obtain a sample stock solution. Pipette 0.6ml of sample stock solution, add diluent to dilute to 10ml, and mix to obtain sample solution. Use the diluent as a blank sample.

Further, the concentration of DL-aminocaprolactam in the above sensitivity solution is 0.124 μg/ml, the concentration of leucine is 0.121 μg/ml, the concentration of phenylalanine is 0.120 μg/ml, and the concentration of threonine is 0.117 μg /ml.

As analyzed in the background technology of this application, the prior art usually conducts a limit detection of one amino acid impurity in the special solvent for adenosylmethionine succinate for injection, but cannot quantitatively detect other amino acid impurities, which leads to inability to Effective quality control of other amino acid impurities.

As analyzed in the background technology of this application, there is no technology in the prior art that can efficiently detect amino acid impurities in the special solvent for adenosylmethionine succinate for injection by a single method. The detection method of amino acid impurities in the special solvent for adenosylmethionine disulfonate, the detection method includes: preparing a standard solution and a sensitivity solution; preparing a sample to be tested, taking the special solvent for adenosylmethionine butanedisulfonate for injection and diluting it with a diluent Dissolving to form a solution; the sample to be detected is detected by high performance liquid chromatography-mass spectrometry, and the mass chromatogram of the standard product of high performance liquid chromatography tandem mass spectrometry of amino acid impurities and the sample is obtained, according to the analysis of the mass chromatogram Results Determine the characteristic ion peaks of various amino acid impurities in the mass chromatogram, and calculate the mass content of each amino acid impurity based on the peak area of the characteristic ion peaks in the sample.

In this application, when the high-performance liquid chromatography and mass spectrometry are used to test the special solvent for adenosylmethionine succinate for injection, the high performance liquid chromatography is used to separate the impurities in the special solvent for adenosylmethionine succinate for injection. Then mass spectrometry carries out further detection to isolate and obtains corresponding mass chromatogram, utilizes mass chromatogram to determine the characteristic ion peak of amino acid impurity and amino acid impurity in corresponding chromatogram, according to this characteristic ion peak area and standard product peak area namely The mass content of amino acid impurities can be obtained. It can be seen that, on the basis of realizing the sufficient separation of amino acid impurities by high performance liquid chromatography, the application uses mass spectrometry data to accurately correspond to the characteristic ion peaks of amino acid impurities in the mass chromatogram, thereby obtaining the accurate mass content of amino acid impurities , to provide a reliable data basis for the effective quality control of amino acid impurities.

The establishment of the standard curve in this application can refer to the process of establishing the standard curve commonly used in the liquid chromatography test in the prior art. In one embodiment, the above step S1 uses the solution of the amino acid impurity standard as the standard solution, preferably preparing the standard The solvent of the solution is a diluent, preferably the diluent is a mixture of methanol and water, more preferably the volume ratio of methanol and water is 60:40.

Since amino acid impurities exist as impurities in the special solvent for adenosylmethionine succinate for injection, their content is relatively small. In order to further ensure the accuracy of the test results, the mass content of amino acid impurities in the above-mentioned standard solution is preferably 0.4 μg/mL. The reliability of the standard curve obtained by using the standard solution of the above concentration as the basis of the test is better.

In order to completely dissolve the amino acid impurities in the special solvent for adenosylmethionine for injection as soon as possible, it is preferable to dilute the special solvent for adenosylmethionine for injection with a diluent in the above step S2 to form a sample solution , the preferred diluent is a mixture of methanol and water, more preferably the volume ratio of methanol and water is 60:40.

When performing high-performance liquid chromatography detection on the special solvent for adenosylmethionine succinate for injection, this application has carried out in-depth research on the detection conditions, especially on the premise of using different chromatographic columns, the composition of the mobile phase and the gradient wash method. The experimental research was carried out with the cooperation of off-programming, and the conditions for the detection of high-performance liquid chromatography were finally determined to include: the chromatographic column was a Poroshell 120 HILIC-Z chromatographic column, the column temperature was 30-40°C, and the flow rate was 0.8ml/min; the mobile phase A is a mixed solution of acetonitrile and buffer solution according to the volume ratio of 95:5, and mobile phase B is a mixed solution of acetonitrile and buffer solution according to the volume ratio of 50:50. The solution; The elution mode of mobile phase is gradient elution, and described gradient elution procedure is:

time (min)	Mobile phase A(%)	Mobile phase B(%)
0.0	75	25
13.0	50	50
13.5	10	90

18.0	10	90
18.1	75	25
25.0	75	25

Preferably, the column temperature is 30°C.

Preferably, the injection volume is 2ul.

Under the above conditions, especially the combination of Poroshell 120 HILIC-Z chromatographic column and the above mobile phase A and mobile phase B, the effective separation of amino acid impurities and other impurities is realized, and the mass determination of amino acid impurities is realized. . At the same time, the properties of the above-mentioned mobile phase A and mobile phase B are stable, which further makes the detection result more reliable.

In order to improve the accuracy of the mass spectrometry detection results, the condition setting of the above-mentioned mass spectrometry detection preferably includes: the ion source is an electrospray ionization source (AJS ESI), the mass spectrometry detector is a triple quadrupole detector (QQQ), and the drying gas temperature is 300 ~350°C, drying gas flow rate is 9~11L/min, atomizing gas pressure is 50~55psi, sheath flow gas temperature is 360~390°C, sheath flow flow rate is 10~12L/min, capillary terminal voltage is 3000~4000V, The nozzle voltage was 450-550V, and the scanning mode was mass spectrometry multiple reaction monitoring (MRM).

In a preferred embodiment, the condition settings for the above-mentioned mass spectrometry detection include: the ion source is an electrospray ion source (AJS ESI), the mass spectrometer detector is a triple quadrupole detector (QQQ), and the drying gas temperature is 350°C , the drying gas flow rate is 11L/min, the atomizing gas pressure is 55psi, the sheath gas temperature is 390°C, the sheath gas flow rate is 12L/min, the capillary terminal voltage is +3500V, the nozzle voltage is +500V, and the scanning mode is mass spectrometry multiple reaction Detection (MRM), the parameters are:

Compared with the existing method for detecting impurities in the special solvent for adenosylmethionine succinate for injection, the present invention changes the cumbersome detection steps of thin-layer chromatography detection, avoids the use of organic and toxic developing agents and chromogenic agents, and is beneficial to For researchers, it is a more healthy method, and at the same time, it can effectively control the amount of multiple amino acid impurities. It solves the problem that amino acid impurities in the special solvent for adenosylmethionine succinate for injection in the prior art cannot be effectively quantified, and can detect each amino acid in the special solvent for adenosylmethionine succinate for injection by one method content of impurities.

The special solvent for adenosylmethionine succinate for injection of the present invention is a sterilized solution of L-lysine, and each (5ml) containing L-lysine (C ₆ H ₁₄ N ₂ O ₂ ) should be The chemical structure of L-lysine is as follows:

Description of drawings

Fig. 1 is the total ion chromatogram of amino acid impurities in the standard solution;

Fig. 2 is the multiple reaction monitoring extracted ion chromatogram of DL-aminocaprolactam in the standard solution;

Fig. 3 is the MRM extraction ion chromatogram of leucine in standard solution;

Fig. 4 is the MRM extraction ion chromatogram of phenylalanine in the standard solution;

Fig. 5 is the MRM extraction ion chromatogram of threonine in the standard solution;

Fig. 6 is the total ion chromatogram of amino acid impurities in the sample solution;

Fig. 7 is the MRM extraction ion chromatogram of DL-aminocaprolactam in the sample solution;

Figure 8 is an MRM-extracted ion chromatogram of leucine in the sample solution;

Fig. 9 is the multiple reaction monitoring extracted ion chromatogram of phenylalanine in the sample solution;

Figure 10 is the multiple reaction monitoring extracted ion chromatogram of threonine in the sample solution;

Fig. 11 is DL-amino caprolactam standard curve;

Figure 12 is a leucine standard curve;

Fig. 13 is phenylalanine standard curve;

Figure 14 is a threonine standard curve.

Detailed ways

It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The following examples illustrate the invention in detail.

The instruments and reagents used in the following tests are as follows.

instrument

Agilent HPLC1260-MS QQQ 6460

Reagent

Methanol, chromatography grade;

Formic acid, mass spec grade;

Acetonitrile, chromatography grade;

Ammonium formate, chromatography grade;

purified water.

The special solvent for adenosylmethionine butyl disulfonate for injection is provided by Zhejiang Hisun Pharmaceutical Co., Ltd.

Example 1

The detection conditions of the high-performance liquid chromatography-mass spectrometer involved in the following detection are:

The conditions for high-performance liquid chromatography detection are set as follows: the chromatographic column is a Poroshell 120 HILIC-Z chromatographic column, the injection volume is 2ul, the column temperature is 30°C, the flow rate is 0.8ml/min, and the mobile phase A is acetonitrile and buffer solution according to The mixed solution of volume ratio 95:5, mobile phase B is the mixed solution of acetonitrile and buffer solution according to volume ratio 50:50, and described buffer solution is the solution that 10mM ammonium formate aqueous solution adjusts pH to 3.0 with formic acid;

The elution mode of mobile phase is gradient elution, and described gradient elution procedure is:

time (min)	A(%)	B(%)
0.0	75	25
13.0	50	50
13.5	10	90
18.0	10	90
18.1	75	25
25.0	75	25

The conditions for mass spectrometry detection were set as follows: the ion source was an electrospray ionization source (AJS ESI), the mass spectrometer detector was a triple quadrupole detector (QQQ), the drying gas temperature was 350 °C, the drying gas flow rate was 11 L/min, and the fog The gas pressure is 55psi, the sheath gas temperature is 390°C, the sheath gas flow rate is 12L/min, the capillary end voltage is +3500V, the nozzle voltage is +500V, the scan mode is MRM, and the parameters are:

System Suitability Establishment Process

The standard solution preparation process is as follows:

The standard solution is the system suitability solution: use a diluent to prepare 5 mg each of phenylalanine, leucine, and threonine, and 6.4 mg of DL-aminocaprolactam hydrochloride (equivalent to 5 mg of DL-aminocaprolactam). A mixed standard solution with a concentration of 0.4 μg/ml was prepared.

The sensitivity solution preparation process is as follows:

Precisely pipette 3.0ml standard solution, add diluent to dilute to 10ml, and mix well.

The sample solution preparation process is as follows:

Take 1 bottle of special solvent for ademethionine succinate for injection (specification 5ml: 428mg), pipette 0.4ml and dilute to 10ml with a diluent, and mix well to obtain a sample stock solution. Pipette 0.6ml of sample stock solution, add diluent to dilute to 10ml, and mix to obtain sample solution.

The detection conditions of the sample solution are the same as those of the standard solution.

The detection process of carrying out high performance liquid chromatography tandem mass spectrometry to standard solution is set as follows:

Add mobile phase and diluent, check for baseline and drift, until the system is stable. Inject a needle of sensitivity solution, the signal-to-noise ratio of each standard peak should not be less than 10. Then inject 6 needles of standard solution continuously, and the RSD of the peak area of each standard should be no more than 10%. The retention time of each impurity is as follows:

Standard	DL-Aminocaprolactam	Leucine	Phenylalanine	threonine
retention time (min)	6.882±1	5.684±1	5.368±1	9.887±1

In order to evaluate whether the system is stable during the entire sequence operation, inject no more than 8 needles of sample solution and after the sequence is completed, inject a needle of standard solution as the central control standard solution. Calculate the RSD of the peak area of each standard in the central control standard solution and the initial 6-shot continuous injection standard solution, and the RSD should not be greater than 10%. (Note: If necessary, a shot of diluent can be properly injected to clean the pipeline residue).

The calculation process of amino acid impurities in the sample solution is as follows:

Calculate the content of phenylalanine, leucine, DL-aminocaprolactam, and threonine in the sample according to the impurity calculation formula below:

in:

A _Spl = the peak area of each amino acid in the sample solution;

A _Std = the average peak area of each standard product in 6 needle standard product solutions;

W _Std = the weighing amount of each standard in the standard solution, expressed in mg;

V _Std = the dilution volume of each standard in the standard solution, expressed in ml;

D = sample dilution factor;

P = the purity of each standard;

Lc = labeled amount of adenosylmethionine dimethylsulfonate special solvent for injection, which is 428mg/5ml = 85.6mg/ml.

Accuracy recovery and precision verification were detected by addition method.

Accuracy stock solution: Weigh 4.822mg of phenylalanine, 5.040mg of leucine, 4.898mg of threonine, and 6.669mg of DL-aminocaprolactam hydrochloride in a 25ml volumetric flask, dissolve with diluent and Dilute to the mark and mix to obtain stock solution A. Precisely pipette 0.4ml of stock solution A into a 10ml volumetric flask, dilute to the mark with diluent, and mix well to obtain the accuracy stock solution.

Sample stock solution: Take 1 bottle of special solvent for adenosylmethionine succinate for injection (specification 5ml: 428mg), pipette 0.4ml and dilute to 10ml with diluent, and mix well to obtain the sample stock solution

30% accuracy sample solution: take 0.15ml accuracy stock solution and 0.6ml sample stock solution, dilute to 10ml with diluent, mix well, and get ready. Prepare 3 copies in parallel

100% accuracy sample solution: take 0.5ml accuracy stock solution and 0.6ml sample stock solution, dilute to 10ml with diluent, mix well, and get ready. Prepare 3 copies in parallel.

200% accuracy sample solution: take 1.0ml accuracy stock solution and 0.6ml sample stock solution, dilute to 10ml with diluent, mix well, and get ready. Prepare 3 copies in parallel.

Acceptance standard: the average recovery rate of three samples of each concentration is 70%-130%. (Refer to the Chinese Pharmacopoeia Analytical Method Validation Regulations)

Inject flow and diluent, check for baseline and drift, until the system is stable. Inject a needle of sensitivity solution, the signal-to-noise ratio of each standard peak should not be less than 10. Then inject 6 needles of standard solution continuously, the RSD of the peak area of each standard should be no more than 10%, inject 1 needle for each sample solution, and inject no more than 8 needles of sample solution and one needle of central control standard at the end of the sequence solution (standard solution). According to the concentration and mass chromatogram of the standard solution, the detected amount, recovery rate and average recovery rate of each concentration of non-volatile extracts were calculated.

The calculation results are shown in Table 1-Table 4 below.

Table 1. DL-aminocaprolactam accuracy test results

Table 2. Experimental results of leucine accuracy

Table 3. Experimental results of phenylalanine accuracy

Table 4. Experimental results of threonine accuracy

linearity verification

Preparation of standard solution

Weigh 5.001mg of phenylalanine, 5.033mg of leucine, 4.883mg of threonine, 6.662mg of DL-aminocaprolactam hydrochloride and place them in a 25ml volumetric flask, dissolve them with a diluent and adjust the volume to the mark. Mix well to obtain standard stock solution A. Precisely pipette 0.4ml of stock solution A into a 10ml volumetric flask, dilute to the mark with diluent, and mix well to obtain standard stock solution B. Precisely pipette 0.5ml of stock solution B into a 10ml volumetric flask, dilute to the mark with a diluent, and mix well to obtain a concentration of phenylalanine, leucine, threonine, and DL-aminocaprolactam of about 0.4μg/ml Standard solution.

Preparation of linear stock solutions

The linear stock solution is the same as the standard stock solution B above.

Preparation of linear solutions

Prepare a linear solution using the linear stock solution, see Table 5 for details.

Table 5. Preparation of linear solutions

Accreditation standard

The correlation coefficient (R ² ) is not less than 0.98

The linearity results of DL-aminocaprolactam, leucine, phenylalanine and threonine are shown in Table 6-Table 9, Figure 11-Figure 14.

Table 6. DL-aminocaprolactam linearity test results

Table 7. Leucine linearity test results

Table 8. Results of phenylalanine linearity experiment

Table 9. Threonine linearity experiment results

Exclusive verification

Preparation of standard solution

Weigh 5.001mg of phenylalanine, 5.033mg of leucine, 4.883mg of threonine, 6.662mg of DL-aminocaprolactam hydrochloride and place them in a 25ml volumetric flask, dissolve them with a diluent and adjust the volume to the mark. Mix well to obtain standard stock solution A. Precisely pipette 0.4ml of stock solution A into a 10ml volumetric flask, dilute to the mark with diluent, and mix the standard to obtain stock solution B. Precisely pipette 0.5ml of stock solution B into a 10ml volumetric flask, dilute to the mark with a diluent, and mix well to obtain a concentration of phenylalanine, leucine, threonine, and DL-aminocaprolactam of about 0.4μg/ml Standard solution.

Preparation of phenylalanine standard solution

Weigh 4.938mg of phenylalanine and place it in a 25ml volumetric flask, dissolve it with a diluent and set the volume to the mark, and mix well. Precisely pipette 20 μl of the above solution into a 10ml volumetric flask, dilute to the mark with a diluent, and mix well to obtain a phenylalanine standard solution with a concentration of about 0.4 μg/ml.

Preparation of leucine standard solution

Weigh 4.900mg of leucine and place it in a 25ml volumetric flask, dissolve it with a diluent and set the volume to the mark, and mix well. Precisely pipette 20 μl of the above solution into a 10ml volumetric flask, dilute to the mark with a diluent, and mix well to obtain a leucine standard solution with a concentration of about 0.4 μg/ml.

Preparation of DL-aminocaprolactam standard solution

Weigh 6.477mg of DL-aminocaprolactam hydrochloride and place it in a 25ml volumetric flask, dissolve it with diluent and set the volume to the mark, and mix well. Precisely pipette 20 μl of the above solution into a 10ml volumetric flask, dilute to the mark with a diluent, and mix well to obtain a DL-aminocaprolactam standard solution with a concentration of about 0.4 μg/ml.

Preparation of threonine standard solution

Weigh 4.868mg of threonine and place it in a 25ml volumetric flask, dissolve it with a diluent and set the volume to the mark, and mix well. Precisely pipette 20 μl of the above solution into a 10ml volumetric flask, dilute to the mark with a diluent, and mix well to obtain a threonine standard solution with a concentration of about 0.4 μg/ml.

Preparation of sample stock solutions

Take 1 bottle of special solvent for ademethionine succinate for injection (specification 5ml: 428mg), pipette 0.4ml and dilute to 10ml with a diluent, and mix well to obtain a sample stock solution.

Preparation of blank sample solution

Pipette 0.6ml of the above sample stock solution, add diluent to dilute to 10ml, mix well, and obtain.

Preparation of spiked sample solution

Take 0.5ml of the above standard stock solution B and 0.6ml of the sample stock solution, dilute to 10ml with diluent, mix well, and obtain.

Accreditation standard

The interference of the blank solvent (mobile phase, diluent) in the retention time window of each amino acid shall not be greater than 10% of each standard solution. (Take certain measures when necessary)

The specificity results are detailed in Table 10.

Table 10. Specificity experiment results

Limit of detection and limit of quantitation verification

(LOQ) limit of quantitation solution:

Standard solution: Weigh 5.001mg of phenylalanine, 5.033mg of leucine, 4.883mg of threonine, and 6.662mg of DL-aminocaprolactam hydrochloride in a 25ml volumetric flask, dissolve and set Make up to the mark and mix well to obtain stock solution A. Precisely pipette 0.4ml of stock solution A into a 10ml volumetric flask, dilute to the mark with diluent, and mix well to obtain stock solution B. Precisely pipette 0.5ml of stock solution B into a 10ml volumetric flask, dilute to the mark with a diluent, and mix well to obtain a concentration of phenylalanine, leucine, threonine, and DL-aminocaprolactam of about 0.4μg/ml Standard solution

Pipette 3.0ml of the above standard solution, add diluent to dilute to 10ml, mix well, and use it as the limit of quantitation solution.

(LOD) limit of detection solution: pipette 1.5ml of the above standard solution, add diluent to dilute to 10ml, mix well, and use it as the limit of detection solution.

Accreditation standard:

The signal-to-noise ratio of each amino acid impurity in the 6-pin LOQ solution is ≥10;

The RSDs of the peak areas of amino acid impurities in the 6-pin LOQ solutions were all ≤15%;

The signal-to-noise ratios of amino acid impurities in the 3-shot LOD solutions were all ≥3.

The characteristic ion peak area and signal-to-noise ratio results of the mass chromatogram of the solution are shown in Table 11-Table 12.

Table 11. LOQ experimental results

Table 12. LOD experiment results

Determination process of amino acid impurities in the special solvent for adenosylmethionine succinate for injection:

Sample solution: Take 1 bottle of special solvent for adenosylmethionine succinate for injection (specification 5ml: 428mg), pipette 0.4ml and dilute to 10ml with a diluent, and mix well to obtain a sample stock solution. Pipette 0.6ml of sample stock solution, add diluent to dilute to 10ml, and mix to obtain sample solution. The diluent is a mixed solvent of methanol and purified water at a volume ratio of 60:40. Sample solutions were prepared in parallel in duplicate.

Chromatographic analysis procedure: After the chromatographic system of the instrument is balanced, inject a needle of sensitivity solution to check the signal-to-noise ratio, which is required to be not less than 10. Inject 6 needles of standard solution continuously, and calculate the average value and RSD of the peak areas of the 6 chromatograms of standard solution should not be greater than 10%; inject 1 needle for each sample solution. Inject no more than 8 needles and at the end of the sequence, respectively inject a needle of system adaptability solution. Compared with the first 6 needles of system adaptability solution continuously injected, the RSD of the peak area of amino acid impurities should not be greater than 10%.

The mass content of each impurity is obtained by using the external standard method and according to the mass chromatogram of each impurity of amino acid and the concentration and mass chromatogram of the standard solution, according to the above impurity calculation formula.

See Table 13-Table 14 for system adaptability and sample test results, and see Figure 1-Figure 10 for typical spectra.

Table 13. System adaptability experiment results

Table 14. Sample test results

*Note: ND means that the detection result is less than LOD. The LODs of the four impurities are all 0.03%.

From the above description, it can be seen that the above-mentioned embodiments of the present invention have achieved the following technical effects:

This application has established a high-performance liquid chromatography-mass spectrometry method for tandem testing of amino acids in the special solvent for adenosylmethionine succinate for injection. A unique chromatographic condition was adopted, including the components of the mobile phase and Separation and chromatographic procedures to separate the amino acid impurities in the special solvent for adenosylmethionine succinate for injection, and then further detect the isolates corresponding to each characteristic peak by mass spectrometry to obtain the corresponding mass chromatogram. The figure can determine the amino acid impurities and the characteristic ion peaks of the amino acid impurities in the corresponding chromatogram. According to the characteristic ion peak area and the peak area of the standard product, the mass content of the amino acid impurities can be calculated according to the external standard method. It can be seen that, on the basis of realizing the sufficient separation of amino acid impurities by high performance liquid chromatography, the application uses mass spectrometry data to accurately correspond to the characteristic ion peaks of amino acid impurities in the mass chromatogram, thereby obtaining the accurate mass content of amino acid impurities , to provide a reliable data basis for the effective quality control of amino acid impurities.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (6)

1. A method for detecting amino acid impurities in a special solvent for adenosylmethionine succinate for injection, characterized in that it comprises:

   Step S1: Dissolving the standard substances of DL-aminocaprolactam, phenylalanine, leucine, and threonine in a diluent to obtain a standard solution;

   Step S2: dissolving the special solvent for adenosylmethionine succinate for injection with a diluent to obtain a sample solution;

   Step S3: using high performance liquid chromatography-mass spectrometry to detect the standard solution obtained in step S1 and the sample solution obtained in step S2;

   Wherein, the detection condition of high performance liquid chromatography described in step S3 comprises:

   The chromatographic column is a Poroshell 120 HILIC-Z chromatographic column, the column temperature is 30-40°C, and the flow rate is 0.8ml/min;

   Mobile phase A is acetonitrile and buffer solution according to volume ratio 95:5 mixed solution, mobile phase B is acetonitrile and buffer solution according to volume ratio 50:50 mixed solution, and described buffer solution is 10mM ammonium formate aqueous solution and adjusts pH with formic acid to a solution of 3.0;

   The elution mode of mobile phase is gradient elution, and described gradient elution program is:

   time (min) Mobile phase A(%) Mobile phase B(%) 0.0 75 25 13.0 50 50 13.5 10 90 18.0 10 90 18.1 75 25 25.0 75 25

   The diluent described in step S1 and step S2 is a mixed liquid of methanol and water in a volume ratio of 60:40.
2. The detection method according to claim 1, wherein the column temperature is 30°C.
3. The detection method according to claim 2 or 3, characterized in that the detection conditions of the high performance liquid chromatography further include: the injection volume is 2ul.
4. The detection method according to any one of claims 1-3, wherein the detection conditions of the mass spectrum in step S3 include:

   The ion source is an electrospray ionization source (AJS ESI), the mass spectrometry detector is a triple quadrupole detector (QQQ), the drying gas temperature is 300-350°C, the drying gas flow rate is 9-11L/min, and the atomizing gas pressure is 50-55psi, sheath gas temperature is 360-390°C, sheath gas flow rate is 10-12L/min, capillary terminal voltage is 3000-4000V, nozzle voltage is 450-550V, and the scanning mode is mass spectrometry multiple reaction detection (MRM).
5. The detection method according to any one of claims 1-4, wherein the detection conditions of the mass spectrum in step S3 include:

   The ion source is an electrospray ion source (AJS ESI), the mass spectrometer detector is a triple quadrupole detector (QQQ), the drying gas temperature is 350°C, the drying gas flow rate is 11L/min, the nebulizing gas pressure is 55psi, and the sheath flow gas The temperature is 390°C, the sheath flow rate is 12L/min, the capillary terminal voltage is +3500V, the nozzle voltage is +500V, and the scanning mode is mass spectrometry multiple reaction monitoring (MRM).
6. The detection method according to claim 5 or 4, wherein the parameters of the mass spectrometry multiple reaction detection are:

   

Images