CN112697930A - Method for detecting related substance 2-sulfotryptophan in compound amino acid injection - Google Patents
Method for detecting related substance 2-sulfotryptophan in compound amino acid injection Download PDFInfo
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Abstract
The invention discloses a method for detecting related substance 2-sulfotryptophan in a compound amino acid injection. In one aspect, the invention relates to compounds of formula I. In another aspect, the invention also relates to the use of the compound of formula I in the examination of related substances in compound amino acid injection, wherein the compound amino acid injection comprises tryptophan and sodium bisulfite or sodium metabisulfite; for example, the compound amino acid injection is selected from: compound amino acid (15), dipeptide (2), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II) and compound amino acid injection (18 AA-III). In another aspect, the method for detecting related substances in the compound amino acid injection comprises the step of measuring the content of the impurity 2-sulfotryptophan in the injection by using a high performance liquid chromatography. The method of the invention can rapidly and accurately determine the content of relevant impurities in the injections.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a method for detecting compound amino acid injection, and particularly relates to a method for detecting a related substance 2-sulfotryptophan in the compound amino acid injection. The method of the present invention exhibits excellent technical effects as described in the present invention.
Background
The compound amino acid injection is a sterilized aqueous solution prepared from a plurality of amino acids, is mainly used for patients who can not take orally or supply nutrition through intestinal tracts and can not meet the requirement of nutrition, and meets the requirement of an organism for obtaining amino acid synthetic protein through intravenous infusion. In the formula of the product, besides amino acid components, sodium bisulfite or sodium metabisulfite with a certain concentration is added as an antioxidant to inhibit impurities generated by oxidation reaction of part of easily-oxidizable amino acids in the product.
Unfortunately, however, it has been found that certain complex amino acid injections have an unknown impurity that is typical and may be associated with tryptophan, and it would be highly desirable to those skilled in the art to track the source of such impurities and effectively detect them.
Disclosure of Invention
The invention aims to provide a method for detecting related substances in compound amino acid injection and also relates to an impurity compound for detecting by the method. It has been surprisingly found that tryptophan related impurities in compound amino acid injection can be effectively detected by the method of the invention. The present invention has been completed based on such findings.
To this end, the invention provides, in a first aspect, the use of a compound of formula I below in the examination of substances related to compound amino acid injection:
The use according to the first aspect of the invention, wherein the compound amino acid injection comprises tryptophan and sodium bisulfite or sodium metabisulfite.
The use according to the first aspect of the present invention, wherein the compound amino acid injection is selected from the group consisting of: compound amino acid (15), dipeptide (2), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II) and compound amino acid injection (18 AA-III). These amino acid injections have been loaded into the Chinese pharmacopoeia, and for example, the above 5 injections have been described in the "pharmacopoeia of the people's republic of China" 2020 edition on pages 977, 978, 979, 981 and 982, respectively; in the context of the present invention, reference to the above-mentioned various injections, unless otherwise specified, is intended to refer to commercially available samples of the species, which, if specifically indicated as home-made samples, are prepared by themselves according to the above-mentioned pharmacopoeia formulation; wherein the compound amino acid injection (18AA) is a product with the concentration of 12 percent, and the compound amino acid injection (18AA-II) is a product with the concentration of 5 percent. Typically, these compound amino acid injections contain tryptophan and sodium bisulfite or sodium metabisulfite.
Further, the second aspect of the present invention relates to a method for detecting related substances in compound amino acid injection, wherein the method uses high performance liquid chromatography to determine the content of the impurity 2-sulfotryptophan in the injection.
The method according to the second aspect of the present invention, wherein the high performance liquid chromatography is performed according to the specifications in the four-part general regulation 0512 of the 2020 edition of the Chinese pharmacopoeia.
The method according to the second aspect of the invention, comprising the operations of:
according to the specification of high performance liquid chromatography in the general rules 0512 of four departments in 2020 edition of Chinese pharmacopoeia;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, ammonium sulfate buffer solution (5 g of ammonium sulfate and 5.25g of sodium heptanesulfonate are taken and 900ml of water is added for dissolution), the pH value is adjusted to 1.5 by hydrochloric acid and 1000ml of water is added), acetonitrile (97:3) is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is carried out according to the following table, the sample introduction amount is 25 mu l, the column temperature is 40 ℃, and the detection wavelength is 220 nm;
| Time | B％ |
| 0 | 0 |
| 18 | 0 |
| 27 | 45 |
| 32 | 0 |
| 53 | 0 |
control solution: precisely weighing a proper amount of a 2-sulfotryptophan reference substance, adding water to dissolve the reference substance, and quantitatively diluting the reference substance to prepare a solution with the concentration of 0.05-0.2 mu g/ml as a reference substance solution; (the concentration of the reference solution can be properly adjusted according to the impurity 2-sulfotryptophan in different test samples, so that the concentration of the reference solution and the concentration of the impurity in the test sample solution are in a corresponding concentration range); test solution: precisely measuring 2.5ml of compound amino acid injection, placing the compound amino acid injection into a 5ml measuring flask, precisely adding 0.2ml of ethylenediamine and 2ml of acetone, uniformly mixing, adding acetone to a constant volume to scale, carrying out vortex mixing for 15min, filtering, precisely measuring 2ml of subsequent filtrate, placing the subsequent filtrate into a 10ml measuring flask, and diluting the subsequent filtrate to scale by using ultrapure water to obtain a sample solution; and (3) testing the applicability of the system: adding a proper amount of a reference substance into the compound amino acid injection until the concentration of the reference substance is 1 mu g/ml to obtain a system applicability test solution, respectively injecting the reference substance solution and the system applicability test solution into a liquid chromatograph, and determining the retention time of 2-sulfotryptophan according to a chromatogram of the reference substance solution, wherein the separation degree between the 2-sulfotryptophan and an adjacent chromatographic peak in the chromatogram of the system applicability test solution is more than 5.0;
the determination method comprises the following steps: precisely measuring a test solution and a reference solution, respectively injecting into a chromatograph, recording a chromatogram, calculating the content of the impurity 2-sulfotryptophan in the test solution by peak area according to an external standard method, and calculating the content of the impurity 2-sulfotryptophan in the compound amino acid injection according to the dilution multiple of a preparation solution.
The method according to the second aspect of the present invention, wherein the size of the column in which octadecylsilane-bonded silica is used as a filler is 4.6X 150mm, 3.5. mu.m.
The method according to the second aspect of the invention, wherein the correlation coefficient r of the linear equation is >0.999 within the concentration range of 0.05. mu.g/ml to 2.0. mu.g/ml for 2-sulfotryptophan.
The process according to the second aspect of the invention, wherein the process recovery of 2-sulfotryptophan is more than 95%, in particular more than 98%, for example between 98% and 102.
The method according to the second aspect of the invention, wherein the limit of detection of 2-sulfotryptophan is less than 0.02 μ g/ml, such as less than 0.015 μ g/ml.
The method according to the second aspect of the present invention, wherein the RSD of the 2-sulfotryptophan content measurement result of the prepared test solution is less than 1%, particularly less than 0.5%, particularly less than 0.2%, within 0 to 24 hours of standing at room temperature.
The method according to the second aspect of the present invention, wherein the compound amino acid injection comprises tryptophan and sodium bisulfite or sodium metabisulfite.
The method according to the second aspect of the present invention, wherein the compound amino acid injection is selected from the group consisting of: compound amino acid (15), dipeptide (2), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II) and compound amino acid injection (18 AA-III).
Further, a third aspect of the present invention provides a compound having a chemical structure represented by formula I below:
in the method steps described herein, although the specific steps described are distinguished in some detail or language from the steps described in the preparation examples of the detailed description section below, those skilled in the art can, nevertheless, fully summarize the above-described method steps in light of the detailed disclosure throughout the present application.
Any embodiment of any aspect of the present application may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the present application, any feature may be applicable to that feature in other embodiments, as long as they do not contradict.
The present application is further described below. All documents cited in this application are incorporated herein by reference in their entirety and to the extent that the meaning of such documents is inconsistent with this application, the express disclosure of this application controls. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even so, it is intended that the present application be more fully described and interpreted herein, to the extent that such terms and phrases are not inconsistent with this known meaning and from the context in which such terms and phrases are expressed.
In the literature (high purity, etc., electronic journal of tumor metabolism and nutrition, 2019-02), attention points of compound amino acid injection and clinical application thereof are described in detail, for example, amino acid is a basic substance constituting protein required by human nutrition, and is also a precursor of other molecules such as antibodies, hormones, enzymes and the like synthesized by the body, and participates in a series of metabolic reactions in the body, can be converted into carbohydrates and fat, and is finally metabolized into carbon dioxide, water and urea to generate energy, so that the compound amino acid injection is one of three macronutrients for nutrition treatment. The proteins constituting the human body are composed of 20 amino acids, 8 of which are not synthesized by the human body itself and must be supplemented from an external source, called Essential Amino Acids (EAA). Amino acids, which are synthesized in the body from precursor materials and do not require access to food, are called non-essential amino acids (NEAA). In addition, some amino acids, which are synthesized in humans but do not meet normal requirements in special cases, are called semi-essential amino acids or Conditionally Essential Amino Acids (CEAA), such as in infants during growth, histidine is an essential amino acid; tyrosine is an essential amino acid for premature infants, cysteine for premature infants and term infants; for patients with renal disease, tyrosine is an essential amino acid; cysteine is an essential amino acid for patients with liver disease. This classification is only from the point of view of in vivo synthesis, not from the point of view of nutritional value. All amino acids are required for the completion of metabolic functions, and each amino acid has its specific physiological function. Amino acids are further classified according to their side chain structure: aromatic amino acids, aliphatic amino acids, and heterocyclic amino acids. Aromatic amino acids include phenylalanine, tryptophan, and tyrosine. Among aliphatic amino acids, leucine, isoleucine and valine have only hydrocarbon chains as side chains and have a branched chain, and thus are called branched-chain amino acids (BCAAs). It is noted that if sufficient utilization of amino acids is to be ensured, it is provided that sufficient non-protein calories (NPC), i.e., calories provided by glucose and fat (about 4kcal for 1g glucose and about 9kcal for 1g fat), are given, otherwise the supplemented amino acids are consumed as calories. The ratio of hot nitrogen in most patients with stable disease is 150kcal to 1g of nitrogen, and the ratio of patients in perioperative period is 100-150 kcal to 1g of nitrogen. The nitrogen content can be calculated by the formula "nitrogen content (g) ═ amino acid content (g) × 16%". The appearance of compound amino acid injection makes it possible to supplement amino acid intravenously, and more than 20 kinds of compound amino acid injection approved by the national drug administration to be on the market exist at present. The compound amino acid injection is mainly divided into a balanced amino acid injection and a disease applicable amino acid injection, and the concentration range of the amino acid is 3-15%. The balanced compound amino acid injection is prepared by taking the mixture ratio of amino acids such as human milk, potatoes and the like as a prescription basis, and is commonly prepared from compound amino acid injection (18AA), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II), compound amino acid injection (18AA-III), compound amino acid injection (18 AA-IV), compound amino acid injection (18 AA-V), compound amino acid injection (14AA), compound amino acid injection (17AA) and the like. The disease-applicable compound amino acid injection is based on the amino acid metabolism characteristics of different diseases, and comprises a liver disease applicable type, a kidney disease applicable type and a wound applicable type. Suitable liver diseases include compound amino acid injection (3AA), compound amino acid injection (6AA), compound amino acid injection (17 AA-III), compound amino acid injection (20AA), etc. Suitable kidney diseases include compound amino acid injection (9AA), compound amino acid injection (18 AA-IX), etc. The wound suitable type comprises compound amino acid injection (15-HBC), compound amino acid injection (18 AA-VII), etc. In addition, the compound amino acid injection special for children, such as the compound amino acid injection for children (18AA-I), the compound amino acid injection for children (18AA-II), the compound amino acid injection for children (19AA-I) and the like. Glutamine is an essential amino acid, and is unstable in aqueous solution and long-term storage and has low solubility (about 3g/L, 20 ℃), so that when the glutamine is used intravenously, the glutamine is added alone to dipeptide alanylglutamine, and the clinically representative drugs are alanylglutamine injection and glycylglutamine and glycyltyrosine dipeptide injection. In order to ensure the stability of amino acids in the compound amino acid injection product, sulfite (sodium metabisulfite and sodium bisulfite) is commonly added in the amino acid preparation as an antioxidant. Sulfites carry a potential risk of toxicity, in two ways, induction of hypersensitivity reactions, damage to tissues and organs. Protein is a key substance for maintaining the survival of an organism, almost all clinical nutrition assessment methods relate to the assessment of protein metabolism, nitrogen balance can dynamically reflect the protein level and the energy balance condition of the organism, and the nitrogen intake amount of the organism is larger than the nitrogen output amount and is positive nitrogen balance, and the nitrogen output amount is smaller than the nitrogen output amount and is negative nitrogen balance. The human body has a regulating effect on nitrogen balance to a certain extent, and when the daily intake of protein by normal adults is increased or decreased, the decomposition rate and the output of protein in the body are increased or decreased. But in certain disease states, such as pancreatitis, post-gastrointestinal resection, cushing's syndrome, trauma, infection, renal dysfunction, burns, etc., negative nitrogen balance can result. Thus, the body also has a different need for proteins, either in the normal state or in different disease states. The compound amino acid injection is a main protein supply form in the current parenteral nutrition, fully meets the protein requirement of organisms, and is expected to achieve the better nutrition treatment purpose by reasonably selecting the amino acid injection with different formulas. The appearance and development of the compound amino acid injection make the intravenous supplement of amino acid possible, but the compound amino acid injection is used as a medicine, and the effectiveness and the safety of the compound amino acid injection in clinical application are ensured. In the aspect of effectiveness, the proportion of various amino acids for intravenous infusion is in accordance with the organism requirements, especially for patients who are in special pathological and physiological states and can not be nourished by the gastrointestinal tract, and the proper amino acid preparation and dosage are selected at proper time according to the metabolic characteristics of special crowds, so that the nutritional treatment effect of the amino acid injection can be achieved. In the aspect of safety, not only the compatibility safety is concerned in clinical application and the known drug interaction is avoided, but also the adverse effects of the components such as antioxidant and the like in the compound amino acid injection on the drug and susceptible people are concerned, and the corresponding quality standard range and the quality progress of the preparation of the compound amino acid injection at home and abroad are determined. In future, with the continuous development of science and technology and pharmaceutical technology, people are expected to apply the safer and more effective compound amino acid injection. Meanwhile, with the continuous development of research in the fields of nutrition metabolism and clinical nutrition, people are expected to provide a more scientific and rigorous clinical application method of the compound amino acid injection.
The method of the present invention exhibits excellent technical effects as described in one or more aspects of the present invention. For example, the content of specific impurities in the compound amino acid injection can be rapidly and accurately determined.
Drawings
FIG. 1: the chromatogram of the test solution of the compound amino acid injection (18AA) is shown in the figure, and the chromatographic peak with the retention time of about 9.2min is the peak of 2-sulfotryptophan.
FIG. 2: the chromatogram of the compound amino acid injection (18AA) without sodium bisulfite shows that the retention time of the chromatogram peak does not have a 2-sulfotryptophan peak at about 9.2 min.
FIG. 3: the chromatogram of the solution obtained by adding sodium bisulfite into the sodium bisulfite-free compound amino acid injection (18AA) shows that the retention time of the chromatogram peak of the 2-sulfotryptophan peak is shown at about 9.2 min.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. The following examples further illustrate the invention without limiting it. Some of the compound amino acid injection samples used in the specific examples of the present invention, if not stated otherwise, are commercially available finished products, and relate to: compound amino acid (15), dipeptide (2), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II) and compound amino acid injection (18AA-III), wherein if the compound amino acid injection is self-prepared injection, the compound amino acid injection or the solution is self-prepared injection according to a prescription for photography.
Example 1: preparation of 2-sulfotryptophan
Dissolving tryptophan 5g and sodium bisulfite 5g in 50ml water, packaging into ampoule bottles, sealing, sterilizing at 115 deg.C under hot pressure for 15min, standing at 40 deg.C for 10 days, filtering, separating with water-acetonitrile (70:30) mixture as mobile phase on Innoval C18 preparative chromatographic column (30 × 250mm, 5 μm, product number IX951530-0) at detection wavelength of 254nm, intercepting the eluate of 2-sulfotryptophan peak (sodium bisulfite does not show chromatographic peak, shows two chromatographic peaks, including tryptophan and impurity), volatilizing solvent, dissolving residue with a little water, adding appropriate amount of cyclohexane to form crystal, and filtering to obtain crystal; recrystallization from water-cyclohexane again gave crystal a, which was identified as 2-sulfotryptophan by this example 2.
Further, with reference to the above method except that sodium hydrogensulfite was replaced with sodium metabisulfite, the crystal B was confirmed to have a structure by the method of example 2, and the result was the same as that of the crystal A, indicating that the impurity obtained by using sodium metabisulfite instead was also 2-sulfotryptophan.
Example 2: structure validation
In example 2, the structure of the crystal A obtained in example 1 was confirmed.
1.1 structural formula, molecular weight
The assay of example 2 finally determined that crystalline a was 2-sulfotryptophan, a compound of formula I:
structural formula (xvi):
the molecular formula is as follows: c11H12N2O5S
Molecular weight: 284.29
1.2 Mass Spectrometry
1.2.1 ESI-MS Mass Spectrometry detection
The instrument model is as follows: mass spectrometer QSTAR Elite LC/MS/MS System (CADM-YQ-014)
An ionization mode: ESI-MS.
The detection basis is as follows: mass spectrometry method of four general rules 0431 in Chinese pharmacopoeia 2015 year edition
And (3) measuring results: the ESI-MS spectrum shows that the [ M + H ] + molecular ion peak of M/z285.1 shows that the relative molecular mass of the product is 284.1.
1.2.2 TOF-MS high resolution Mass Spectrometry detection
And (3) testing conditions are as follows: TOF MS ES (+)/()
Measurement data: 2-sulfotryptophan high resolution mass spectrometry data. The mass-to-charge ratio is detailed in the mass spectrum test data and attribution of table 1.
Table 1: mass spectrometry test data and attribution
| Mass to charge ratio (m/z) | Relative abundance | Molecular formula | Peak assignment | Remarks for note |
| 285.0536 | 100 | C11H12N2O5S | [M+H]+ | Molecular ion peaks of 2-sulfotryptophan |
1.2.3 Mass/Charge ratio analysis
The molecular formula of the 2-tryptophan bisulfite (impurity S) is C11H12N2O5S, the molecular weight calculated in Chemdraw software is 284.29, and the mass-to-nuclear ratio m/e is 284.05; molecular ion peak [ M + H ] actually measured by ESI mass spectrum]+Is m/z 285.1; high resolution mass spectrometric actual determination of [ M + H]+The nucleus ratio (m/z) was 285.0536, giving the molecular formula C with the highest degree of match11H13N2O5S, so the molecular formula is determined as C11H12N2O5S, which corresponds to the formula of 2-hydroxytryptophan sulfite.
And (4) conclusion: the molecular weight of the ESI mass spectrum and the high-resolution mass spectrum is consistent with the calculated value of the impurity S in the Chemdraw software, and the element composition and the molecular formula C are determined11H12N2O5S corresponds to the identified structure.
1.3 nuclear magnetic resonance spectroscopy
The instrument model is as follows: BRUKER AVANCEIII-400 type nuclear magnetic resonance apparatus
Solvent: CD (compact disc)3OD。
1.3.1 nuclear magnetic resonance Spectroscopy Hydrogen Spectroscopy
The following structures are chemical structures marked with corresponding atom numbers and used for nuclear magnetic resonance spectrum analysis, and the attribution details are shown in the chemical shift and the attribution of hydrogen atoms analyzed by nuclear magnetic resonance hydrogen spectrum in the table 2.
Table 2: nuclear magnetic resonance hydrogen spectrum analysis of chemical shift and attribution of hydrogen atoms
| Chemical shift (ppm) | Number of protons | Peak shape | Attribution | COSY correlation coupling |
| 3.545-3.601 | 1 | dd | 8 | 9-H, itself |
| 3.677-3.727 | 1 | dd | 8 | 9-H, itself |
| 4.220-4.250 | 1 | m | 9 | 8-H |
| 7.055-7.092 | 1 | m | 5 | 6-H、4-H |
| 7.181-7.219 | 1 | m | 6 | 7-H、5-H |
| 7.396-7.416 | 1 | d | 7 | 6-H |
| 7.594-7.614 | 1 | d | 4 | 5-H |
2-sulfotryptophan having the molecular formula C11H12N2O5S, in the structureThe product is only soluble in methanol and water but not in other deuterated reagents such as DMSO, so that when the product is tested by using deuterated methanol, 5 active hydrogens cannot be seen, and only 7 hydrogen signals appear. The structure is confirmed according to the chemical shift value, the integral ratio and the coupling splitting condition in combination with a COSY spectrum, and the analysis attribution of each group of proton peaks is as follows:
delta 3.545-3.601ppm is dd double peak, the integral area is 1 hydrogen, one hydrogen signal in 8-position methylene is subjected to adjacent 9-H position hydrogen coupling and self-coupling splitting, and COSY spectrum shows that the COSY spectrum has mutual coupling relation with the other hydrogen signal in 9-H and self 8 positions.
Delta 3.677-3.727ppm is dd double peak, the integral area is 1 hydrogen, one hydrogen signal in 8-position methylene is subjected to adjacent 9-H position hydrogen coupling and self-coupling splitting, and COSY spectrum shows that the signal has mutual coupling relation with the other hydrogen signal in 9-H position and self-8 position.
The delta 4.220-4.250ppm is m triplet, the integrated area is 1 hydrogen, the peak is a 9-position methine peak, and the COSY spectrum shows that 2 hydrogen signals at 8 positions have mutual coupling relation.
The delta 7.055-7.092ppm is m triplet, the integral area is 1 hydrogen, the signal is a hydrogen signal on a 5-position benzene ring, the signal is split into triplets by 6-position and 4-position coupling, and a COSY spectrum shows that the signal has a mutual coupling relation with the 6-position hydrogen signal and the 4-position hydrogen signal.
The delta 7.181-7.219ppm is m triplet, the integral area is 1 hydrogen, the signal is a hydrogen signal on a 6-position benzene ring, the signal is split into triplet by the coupling of 7-position and 5-position, and a COSY spectrum shows that the signal has a mutual coupling relation with the hydrogen signals of 7-position and 5-position.
Delta 7.396-7.416ppm is a doublet peak, the integral area is 1 hydrogen, the signal is a hydrogen signal on a 7-position benzene ring, the signal is split into the doublet peak by 6-position hydrogen coupling, and a COSY spectrum shows that the 6-position hydrogen signal has a mutual coupling relation with the signal.
Delta 7.594-7.614ppm is a doublet peak, the integral area is 1 hydrogen, the signal is a hydrogen signal on a 4-position benzene ring, the signal is split into the doublet peak by the coupling of 5-position hydrogen, and a COSY spectrum shows that the 5-position hydrogen signal has a mutual coupling relation with the signal. And (4) conclusion: the impurity control product has 7 hydrogen signals in total in the hydrogen spectrum, and 5 active hydrogens are exchanged only by adopting deuterated methanol and cannot be seen, and besides, the structure type, the coupling relation and the number of the impurity control product are consistent with the structure of 2-sulfotryptophan.
1.3.2 carbon Spectroscopy of NMR
The following structures are chemical structures for nmr spectroscopy labeled with corresponding atom numbers. The detailed attribution is shown in the following table 3 nuclear magnetic resonance carbon spectrum, DEPT spectrum, HSQC, HMBC data and attribution
Table 3: nuclear magnetic resonance carbon spectrum, DEPT spectrum, HSQC, HMBC data and attribution
2-Tryptophan sulfite has the molecular formula C11H12N2O5S, which has the same appearance as the carbon spectrum, has a total of 11 carbons in the structure, 1 secondary carbon, 6 tertiary carbons, and 5 quaternary carbons. Combining the C spectrum chemical shift value, the DEPT spectrum and the HSQC spectrum, and analyzing and attributing the spectra as follows:
delta 26.26ppm, secondary carbon suggested by DEPT spectrum, 8-position hydrogen spectrum shown by HSQC spectrum and coupling relation thereof, and the 8-position CH can be presumed2A carbon atom.
Delta 54.81ppm, the DEPT spectrum indicates tertiary carbon, the 9-methine carbon signal, the HSQC spectrum shows that 9-hydrogen spectrum has coupling relation, and the carbon can be presumed to be 9-CH carbon atoms.
Delta 107.60ppm, the DEPT spectrum indicates quaternary carbon, the HSQC spectrum shows no relevant peak, and the position of the peak is deduced to be 3-position carbon atom by the presence of carbon.
Delta 113.25ppm, the DEPT spectrum indicates tertiary carbon, and the DEPT spectrum indicates a 7-methine carbon signal, the HSQC spectrum shows that the 7-hydrogen spectrum has a coupling relation, and the carbon can be presumed to be a 7-CH carbon atom.
Delta 120.31ppm, the DEPT spectrum indicates tertiary carbon, the 4-methine carbon signal, the HSQC spectrum shows that the 4-hydrogen spectrum has a coupling relation, and the carbon can be presumed to be a 4-CH carbon atom.
Delta 121.32ppm, the DEPT spectrum indicates tertiary carbon, 5-methine carbon signal, the HSQC spectrum shows that 5-hydrogen spectrum has coupling relation, and the carbon can be presumed to be 5-CH carbon atom.
Delta 125.03ppm, the DEPT spectrum indicates tertiary carbon, the DEPT spectrum is a 6-position methine carbon signal, the HSQC spectrum shows that 6-position hydrogen spectrum has a coupling relation, and the carbon can be presumed to be a 6-position CH carbon atom.
1.3.3 HMBC mapping
No C-H correlation was directly observed between the 5 quaternary carbon signals in the structure, and the identification by combining the chemical shift values and the HMBC spectra is detailed in the data and attribution of the carbon nuclear magnetic resonance spectra, DEPT spectra, HSQC and HMBC shown in Table 7-3. The detailed analysis is as follows:
delta 107.60ppm, the DEPT spectrum suggests quaternary carbons, the HMBC spectrum shows that this set of carbon signals is associated with 4-, 8-, 9-hydrogen, and in combination with the carbon spectrum chemical shift values, it is reasonably assigned as a 3-quaternary carbon signal.
Delta 128.16ppm, the DEPT spectrum suggests quaternary carbons, the HMBC spectrum shows that this set of carbon signals is associated with 5-, 7-, 8-hydrogen, and in combination with the carbon spectrum chemical shift values, it is reasonably assigned as 3a quaternary carbon signal.
Delta 136.08ppm, the DEPT spectrum suggests quaternary carbons, the HMBC spectrum shows that this set of carbon signals is associated with 4, 6-position hydrogens, which can be reasonably assigned as 7 a-position quaternary carbon signals in combination with the carbon spectrum chemical shift values.
δ 138.12ppm, the DEPT spectrum suggests quaternary carbons, the HMBC spectrum shows that this set of carbon signals is associated with 8-position hydrogen, which in combination with the carbon spectrum chemical shift values can be reasonably assigned as 2-position quaternary carbon signals.
Delta 172.5ppm, the DEPT spectrum suggests quaternary carbons, the HMBC spectrum shows that this set of carbon signals is associated with 8, 9-hydrogen, which, in combination with the carbon spectrum chemical shift values, can be reasonably assigned as 10-quaternary carbon signals.
And (4) conclusion: all carbon spectrum signals are reasonably attributed, and the DEPT and HMBC spectrums are used for confirming the carbon types. In conclusion, the attribution of carbon and hydrogen of the sample crystal A determines that the structure of the sample crystal A is 2-sulfotryptophan.
When the method for determining related substances of the compound amino acid injection is used for determining the tryptophan raw material used in example 1, 2-sulfotryptophan is not detected and is lower than the detection limit, which indicates that the crystal A is generated by the reaction of tryptophan and sodium bisulfite or sodium metabisulfite.
According to the preparation of crystal A of example 1, it can be concluded that it is generated by the sulfonation reaction of tryptophan with sulfite, and the reaction formula is as follows:
according to the invention, 2-sulfotryptophan is a specific impurity generated by the reaction of tryptophan in the compound amino acid injection and antioxidant sodium bisulfite or sodium metabisulfite, does not exist in tryptophan raw materials, and a method for effectively detecting the impurity does not exist in the field at present. In order to effectively control the product quality and ensure the safety and reliability of the product in the shelf life, it is necessary to establish a rapid, simple, sensitive and stable method for controlling the content of 2-sulfotryptophan in the compound amino acid injection.
Example 3: qualitative and quantitative determination of 2-sulfotryptophan in compound amino acid injection
The embodiment aims to provide a qualitative and quantitative determination method for 2-sulfotryptophan in compound amino acid injection, which is expected to have the characteristics of rapidness, simplicity, sensitivity and stability, and comprises the following steps:
1. the instrument comprises the following steps: high performance liquid chromatograph
2. A chromatographic column: octadecylsilane chemically bonded silica (C18 column, 4.6X 150mm specification, 3.5 μm, Waters SunAire brand column in this example, and other chromatography column with the same performance can be used) as filler
3. Chromatographic conditions are as follows:
dissolving in ammonium sulfate buffer (5 g ammonium sulfate, 5.25g sodium heptanesulfonate, 900ml water), adjusting pH to 1.5 with hydrochloric acid, and 1000ml water) acetonitrile (97:3) as mobile phase A, and acetonitrile as mobile phase B, loading 25 μ l, column temperature 40 deg.C, and gradient eluting according to Table 1. The detection wavelength is 220 nm.
Table 1: gradient elution chart
| Time | B％ |
| 0 | 0 |
| 18 | 0 |
| 27 | 45 |
| 32 | 0 |
| 53 | 0 |
4. Preparing a test solution: precisely measuring 2.5ml of the compound amino acid injection, placing the compound amino acid injection into a 5ml measuring flask, precisely adding 0.2ml of ethylenediamine and 2ml of acetone, uniformly mixing, adding acetone to a constant volume to scale, carrying out vortex mixing for 15min, then filtering, precisely measuring 2ml of subsequent filtrate, placing the subsequent filtrate into a 10ml measuring flask, and diluting the subsequent filtrate to scale by using ultrapure water (prepared by a pure water machine, the resistivity is more than or equal to 18.2M omega) to obtain a sample solution. [ if the concentration of the impurity 2-sulfotryptophan in the sample solution is too high, the sample solution can be further quantitatively diluted with water ]
5. Preparing a reference substance solution: an appropriate amount of 2-sulfotryptophan (crystal A, having chromatographic purities of 99.87%, 99.85%, 99.94% at wavelengths of 220nm, 254nm, 280nm, respectively, as determined by the method of the chromatographic conditions of example 3) obtained in example 1 was taken, and ultrapure water was added thereto to prepare control solutions having concentrations of 0.051, 0.103, 0.514, 1.028, 1.542, 2.056. mu.g/ml.
6. And (3) verification of methodology:
6.1, linearity
Preparation of a linear solution: respectively and precisely measuring 25 mul of the serial 2-sulfotryptophan reference substance solutions, injecting into a liquid chromatograph, and recording the chromatogram. Linear regression was performed with the concentration as abscissa and the peak area as ordinate. The data are detailed in table 2 for linear experimental results.
Table 2: 2-sulfotryptophan Linear test results
| Linear solution | 2-sulfotryptophan concentration (. mu.g/ml) | Peak area of 2- |
| 1 | 0.051 | 0.0453 |
| 2 | 0.103 | 0.0906 |
| 3 | 0.514 | 0.4376 |
| 4 | 1.028 | 0.8735 |
| 5 | 1.542 | 1.3351 |
| 6 | 2.056 | 1.7672 |
And (3) test results: the 2-sulfotryptophan is in the range of 0.051 mu g/ml to 2.056 mu g/ml, the linear equation of the concentration of the 2-sulfotryptophan and the peak area is that y is 0.8606 x-0.0012, and the correlation coefficient r is 0.9999; and the test requirements are met.
6.2, accuracy
Accuracy was investigated in terms of recovery.
Precisely measuring 2.5ml of compound amino acid injection (18AA) (batch number JM2000157) and placing the compound amino acid injection into a 5ml measuring flask, precisely adding 0.2ml of ethylenediamine and 2ml of acetone, uniformly mixing, adding acetone to a constant volume to a scale, carrying out vortex mixing for 15min, filtering, precisely measuring 2ml of subsequent filtrate, placing the subsequent filtrate into a 10ml measuring flask, adding 24 mu l, 30 mu l and 36 mu l of stock solution (the content of 2-sulfotryptophan is 0.1028 mu g/ml) for linear test, (n is 3 multiplied by 3), adding water to dilute the subsequent filtrate to the scale, shaking uniformly, injecting samples respectively, recording a chromatogram, and calculating the recovery rate according to an external standard method. The specific results are shown in Table 3.
Table 3: experimental result of sample recovery rate of 2-sulfotryptophan
And (4) conclusion: the recovery rate of the 2-sulfotryptophan is within the range of 99.32-102.04%, the average recovery rate is 101.2%, the RSD is 0.8%, and the test requirements are met.
6.3, precision
Precision was examined by repeatability tests. A sample of the batch number JM2000157 is taken, 6 parts of sample solution are prepared according to the item of '4 preparation of test sample solution', and the sample solution is used as a repetitive sample solution for measurement so as to investigate the precision of the method. The results are detailed in Table 4 for the results of the repeated experiments.
Table 4: results of 2-sulfotryptophan reproducibility test
6.4, quantitation Limit and detection Limit
Preparing a 2-sulfotryptophan quantitative limit and detection limit solution: the solution 1 for linear test (2-sulfotryptophan concentration 0.051 mug/ml) is diluted step by step and then is measured until the signal to noise ratio S/N is taken as 10:1 and is taken as the limit of quantification and the S/N is taken as 3:1 and is taken as the limit of detection.
And (4) conclusion: the limit of 2-sulfotryptophan quantification is 0.026 mug/ml, and the limit of detection is 0.013 mug/ml.
6.5 solution stability
A sample solution prepared from a sample with the batch number of JM2000157 is taken and placed at room temperature, the content of 2-sulfotryptophan is measured at 0 hour, 4 hours, 8 hours, 12 hours and 24 hours respectively, and the stability of the solution is inspected according to the relative standard deviation of the peak area of the 2-sulfotryptophan.
The results are detailed in table 5 for stability test results.
Table 5: 2-sulfotryptophan stability test results
And (4) conclusion: the 2-sulfotryptophan is stable within 24 hours at room temperature and meets the test requirements.
6.6 System suitability test for HPLC method: adding a proper amount of a reference substance into the compound amino acid injection until the concentration of the reference substance is 1 mu g/ml to obtain a system applicability test solution, respectively injecting the reference substance solution and the system applicability test solution into a liquid chromatograph, and determining the retention time of the 2-sulfotryptophan according to a chromatogram of the reference substance solution, wherein the separation degree between the 2-sulfotryptophan and an adjacent chromatographic peak in the chromatogram of the system applicability test solution is more than 5.0. The test results are all more than 8.0.
6.7, content determination method of impurity 2-sulfotryptophan in the injection: precisely measuring a test solution and a reference solution (selecting the reference solution with the concentration equivalent to that of the 2-sulfotryptophan in the test solution), respectively injecting into a chromatograph, recording a chromatogram, calculating the content of the impurity 2-sulfotryptophan in the test solution by peak area according to an external standard method, and calculating the content of the impurity 2-sulfotryptophan in the compound amino acid injection according to the dilution multiple of a preparation solution.
In the method of this example 3, the chromatogram of the sample of Compound amino acid injection (18AA) is shown in FIG. 1, in which the peak of the chromatogram with retention time of about 9.2min is the peak of 2-sulfotryptophan.
In addition, when crystal a and crystal B obtained in example 1 were measured using the chromatographic conditions of example 3, the retention times of the main peaks of both samples were at about 9.2min, and the chromatographic purities calculated by the hundred product normalization method were each greater than 99%.
Example 31: the accuracy test, i.e., recovery test, was performed with reference to the "6.2, accuracy" method of example 3 above, except that no acetone was added when the test solution was prepared, resulting in average recoveries of 87.32%, 91.61%, 88.76% at the low and medium three concentration levels, respectively; the accuracy test, i.e., the recovery test, was performed with reference to the "6.2, accuracy" method of example 3 above, except that in which the test solution was prepared without adding ethylenediamine, the average recoveries were 92.47%, 89.33%, 90.38% at the low and medium three concentration levels, respectively; the accuracy test, i.e., the recovery rate test, was performed by referring to the method of "6.2, accuracy" in example 3, except that the procedure for preparing the test solution was changed to "precisely measure 1ml of compound amino acid injection (18AA), place it in a 10ml measuring flask, add 24 μ l, 30 μ l, and 36 μ l of stock solution for linear test (2-sulfotryptophan content 0.1028 μ g/ml), (n ═ 3 × 3), add water to dilute to the scale, shake it, sample it separately", i.e., neither acetone nor ethylenediamine was added, and as a result, the average recovery rates of the low and medium concentration levels were 88.39%, 87.46%, and 89.27%, respectively; the RSD for these average recovery results for example 31 were all in the range of 0.6% to 1.4%; the results of this example 31 show that the recovery rate is clearly unacceptable when these modified dosing methods are used as an impurity level measurement. Example 32: referring to the formula of compound amino acid injection (18AA) described in Chinese pharmacopoeia but without adding sodium bisulfite, the compound amino acid injection (18AA) without sodium bisulfite is prepared into solution according to a conventional method, then is subpackaged into ampoules and sealed, 5ml of each ampoule is sterilized at 115 ℃ under hot pressure for 15min, and then is placed at 40 ℃ for 10 days, so as to prepare the compound amino acid injection (18AA) without sodium bisulfite, the chromatogram of the compound amino acid injection (18AA) is determined by using the method of example 3, and the chromatogram is shown in figure 2, which shows that the chromatographic peak is not appeared when the retention time is about 9.2min, which indicates that the injection without adding sodium bisulfite does not have the impurity 2-.
Example 33: adding sodium bisulfite (to the concentration of 0.5mg/ml) into the sodium bisulfite-free compound amino acid injection (18AA) which is prepared by the method in the above example 32 and is subjected to sterilization and treatment at 40 ℃, subpackaging the sodium bisulfite into ampoule bottles for sealing by melting, sterilizing under hot pressure at 115 ℃ for 15min, and standing at 40 ℃ for 10 days to obtain an additional sodium bisulfite liquid medicine; the chromatogram of the liquid medicine measured by the method of example 3 is shown in FIG. 3, which shows the chromatographic peak of impurity 2-sulfotryptophan peak at about 9.2min of retention time, and shows the impurity 2-sulfotryptophan peak which is typical of the reaction of tryptophan and sulfite after the addition of sodium bisulfite.
Example 34: referring to the formula of the compound amino acid injection (18AA) recorded in Chinese pharmacopoeia but without adding tryptophan, the compound amino acid injection (18AA) is prepared into a solution according to a conventional method, then the solution is subpackaged into ampoules and sealed, 5ml of the solution is packaged in each ampoule, the autoclave sterilization is carried out at 115 ℃ for 15min, and then the solution is placed at 40 ℃ for 10 days, so that the compound amino acid injection (18AA) without tryptophan is prepared, and the result of the determination by using the method of the example 3 shows that the retention time in a chromatogram (not provided) is no chromatographic peak at about 9.2min, which indicates that the injection without the tryptophan does not have an impurity peak of 2-sulfotryptophan.
Example 4: determination of compound amino acid injectionImpurity content of
The high performance liquid chromatography of embodiment 3 of the present invention is performed according to the specifications of the four-part general regulation 0512 of the 2020 edition of the Chinese pharmacopoeia, and the method can be briefly summarized as follows:
chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (for example, the specification of the used chromatographic column is C18 column, 4.6X 150mm, 3.5 μm, in this case, a Waters SunAire brand chromatographic column can be used, other chromatography columns with the same effect can also be used), ammonium sulfate buffer solution (taking 5g of ammonium sulfate, 5.25g of sodium heptane sulfonate, adding 900ml of water for dissolution, adjusting the pH to 1.5 by hydrochloric acid, adding water to 1000ml), acetonitrile (97:3) as mobile phase A, and acetonitrile as mobile phase B, and performing gradient elution according to the following table, wherein the sample amount is 25 μ l, the column temperature is 40 ℃, and the detection wavelength is 220 nm;
| Time | B％ |
| 0 | 0 |
| 18 | 0 |
| 27 | 45 |
| 32 | 0 |
| 53 | 0 |
control solution: precisely weighing a proper amount of a 2-sulfotryptophan reference substance, adding water to dissolve the reference substance, and quantitatively diluting the reference substance to prepare a solution with the concentration of 0.05-0.2 mu g/ml as a reference substance solution; (the concentration of the reference solution can be properly adjusted according to the impurity 2-sulfotryptophan in different test samples, so that the concentration of the reference solution and the concentration of the impurity in the test sample solution are in a corresponding concentration range); test solution: precisely measuring 2.5ml of compound amino acid injection, placing the compound amino acid injection into a 5ml measuring flask, precisely adding 0.2ml of ethylenediamine and 2ml of acetone, uniformly mixing, adding acetone to a constant volume to scale, carrying out vortex mixing for 15min, filtering, precisely measuring 2ml of subsequent filtrate, placing the subsequent filtrate into a 10ml measuring flask, and diluting the subsequent filtrate to scale by using ultrapure water to obtain a sample solution; and (3) testing the applicability of the system: adding a proper amount of a reference substance into the compound amino acid injection until the concentration of the reference substance is 1 mu g/ml to obtain a system applicability test solution, respectively injecting the reference substance solution and the system applicability test solution into a liquid chromatograph, and determining the retention time of 2-sulfotryptophan according to a chromatogram of the reference substance solution, wherein the separation degree between the 2-sulfotryptophan and an adjacent chromatographic peak in the chromatogram of the system applicability test solution is more than 5.0;
the determination method comprises the following steps: precisely measuring a test solution and a reference solution, respectively injecting into a chromatograph, recording a chromatogram, calculating the content of the impurity 2-sulfotryptophan in the test solution by peak area according to an external standard method, and calculating the content of the impurity 2-sulfotryptophan in the compound amino acid injection according to the dilution multiple of a preparation solution.
By using the HPLC method summarized above, 1 batch of each of five commercially available injections, namely compound amino acid (15) dipeptide (2) injection, compound amino acid injection (18AA), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II) and compound amino acid injection (18AA-III), was used to determine the content of the impurity, 2-sulfotryptophan, in the injections, and the results were 3.36. mu.g/ml, 2.72. mu.g/ml, 5.61. mu.g/ml, 4.31. mu.g/ml and 6.27. mu.g/ml, respectively. In addition, after the 5 injections are placed at the temperature of 40 ℃ for 1 month, the content of the impurity 2-sulfotryptophan in the injections is measured, the content of the impurity in each sample is compared with the result of 0 month to calculate the increase percentage of the impurity, the increase percentage of the impurity 2-sulfotryptophan in the 5 injections is in the range of 67-94%, for example, the increase percentage of the impurity 2-sulfotryptophan in the compound amino acid injection (18AA) is 84.2%.
It will be evident to those skilled in the art that the present application is not limited to the details of the foregoing illustrative embodiments, and that the present application may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. The application of the compound shown in the formula I in the inspection of related substances of the compound amino acid injection or the method for inspecting the compound shown in the formula I in the compound amino acid injection is as follows:
2. the use according to claim 1, wherein the compound amino acid injection comprises tryptophan and sodium bisulfite or sodium metabisulfite; for example, the compound amino acid injection is selected from: compound amino acid (15), dipeptide (2), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II) and compound amino acid injection (18 AA-III).
3. The method for checking related substances in the compound amino acid injection comprises the step of measuring the content of the impurity 2-sulfotryptophan in the injection by using a high performance liquid chromatography.
4. The method according to claim 3, wherein the high performance liquid chromatography is performed according to the specifications in the four-part general regulation 0512 of the Chinese pharmacopoeia 2020 edition.
5. A method according to claim 3, comprising the following operations:
according to the specification of high performance liquid chromatography in the general rules 0512 of four departments in 2020 edition of Chinese pharmacopoeia;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, ammonium sulfate buffer solution (5 g of ammonium sulfate and 5.25g of sodium heptanesulfonate are taken and 900ml of water is added for dissolution), the pH value is adjusted to 1.5 by hydrochloric acid and 1000ml of water is added), acetonitrile (97:3) is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is carried out according to the following table, the sample introduction amount is 25 mu l, the column temperature is 40 ℃, and the detection wavelength is 220 nm;
control solution: precisely weighing a proper amount of a 2-sulfotryptophan reference substance, adding water to dissolve the reference substance, and quantitatively diluting the reference substance to prepare a solution with the concentration of 0.05-0.2 mu g/ml as a reference substance solution; (the concentration of the reference solution can be properly adjusted according to the impurity 2-sulfotryptophan in different test samples, so that the concentration of the reference solution and the concentration of the impurity in the test sample solution are in a corresponding concentration range); test solution: precisely measuring 2.5ml of compound amino acid injection, placing the compound amino acid injection into a 5ml measuring flask, precisely adding 0.2ml of ethylenediamine and 2ml of acetone, uniformly mixing, adding acetone to a constant volume to scale, carrying out vortex mixing for 15min, filtering, precisely measuring 2ml of subsequent filtrate, placing the subsequent filtrate into a 10ml measuring flask, and diluting the subsequent filtrate to scale by using ultrapure water to obtain a sample solution; and (3) testing the applicability of the system: adding a proper amount of a reference substance into the compound amino acid injection until the concentration of the reference substance is 1 mu g/ml to obtain a system applicability test solution, respectively injecting the reference substance solution and the system applicability test solution into a liquid chromatograph, and determining the retention time of 2-sulfotryptophan according to a chromatogram of the reference substance solution, wherein the separation degree between the 2-sulfotryptophan and an adjacent chromatographic peak in the chromatogram of the system applicability test solution is more than 5.0;
the determination method comprises the following steps: precisely measuring a test solution and a reference solution, respectively injecting into a chromatograph, recording a chromatogram, calculating the content of the impurity 2-sulfotryptophan in the test solution by peak area according to an external standard method, and calculating the content of the impurity 2-sulfotryptophan in the compound amino acid injection according to the dilution multiple of a preparation solution.
6. A process as claimed in claim 3, wherein the size of the column packed with octadecylsilane-bonded silica used is 4.6X 150mm, 3.5. mu.m.
7. A method according to claim 3, wherein the correlation coefficient r of the linear equation for 2-sulfotryptophan is >0.999 over a concentration range of 0.05 μ g/ml to 2.0 μ g/ml; alternatively, the process recovery of 2-sulfotryptophan therein is greater than 95%, particularly greater than 98%, for example between 98% and 102.
8. A method according to claim 3, wherein the limit of detection of 2-sulfotryptophan is less than 0.02 μ g/ml, such as less than 0.015 μ g/ml; or the RSD of the content determination result of the 2-sulfotryptophan in the prepared test solution is less than 1%, particularly less than 0.5%, particularly less than 0.2% within 0-24 hours of standing at room temperature.
9. The method according to claim 3, wherein the compound amino acid injection comprises tryptophan and sodium bisulfite or sodium metabisulfite; or, the compound amino acid injection is selected from: compound amino acid (15), dipeptide (2), compound amino acid injection (18AA-I), compound amino acid injection (18AA-II) and compound amino acid injection (18 AA-III).
10. A compound having the chemical structure shown in formula I below:
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