CN110806448B - Method for determining free ammonia in compound amino acid injection - Google Patents
Method for determining free ammonia in compound amino acid injection Download PDFInfo
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- CN110806448B CN110806448B CN201911146174.8A CN201911146174A CN110806448B CN 110806448 B CN110806448 B CN 110806448B CN 201911146174 A CN201911146174 A CN 201911146174A CN 110806448 B CN110806448 B CN 110806448B
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- amino acid
- solution
- free ammonia
- acid injection
- compound amino
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Abstract
The invention discloses a method for measuring free ammonia in compound amino acid injection, which comprises the following steps: providing an amino acid analyzer and a chromatographic column; preparing a buffer solution for separation and analysis; preparing a derivatization reaction reagent; the flow rate of the mobile phase is 0.40ml/min, the flow rate of the derivatization reagent is 0.35ml/min, the column temperature is 57 ℃, the reactor temperature is 135 ℃, the detection wavelength is 570nm, the sample injection amount is 5 mu l, and the elution is carried out according to the gradient; preparing a test solution; preparing a control solution and performing chromatographic determination. And during chromatographic determination, the concentration of free ammonia in the test solution is calculated by peak area according to an external standard method, and the content of the free ammonia in the compound amino acid injection is calculated according to the preparation method of the test solution. Also relates to compound amino acid injection determined by the method. The method of the invention exhibits excellent technical effects as described in the specification, such as rapidness, simplicity, sensitivity, stability.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a detection method of a medicine, and particularly relates to a detection method of a compound amino acid injection, wherein the method is particularly used for determining free ammonia 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.
At present, there are up to 500 approved literature references for domestic drugs related to compound amino acid injection recorded by the State food and drug administration, and 5 drug standards for compound amino acid injection recorded by the second part of the Chinese pharmacopoeia 2015 edition.
During the production and storage of the compound amino acid injection, free ammonia (namely NH) can be generated due to deamination of amino acid3). Because the compound amino acid injection belongs to a large-capacity parenteral nutrition supplement, the use time of a patient is long, the dosage of the patient is large, for the patient who infuses the compound amino acid injection for a long time, a large amount of ingested free ammonia can be converted into nitrite under certain conditions, the nitrite can oxidize low-iron hemoglobin which normally carries oxygen in blood into high-iron hemoglobin to lose the oxygen carrying capacity to cause tissue hypoxia, and the nitrite is possibly converted into a strong carcinogen nitrosamine in vivo, thereby having great harm to the health of the human body. Therefore, the content of free ammonia in the compound amino acid injection product is strictly controlled.
The existing method for measuring free ammonia generally adopts enzyme catalysis ultraviolet-visible spectrophotometry. The principle is as follows: the ammonia and 2-ketoglutarate oxidize Nicotinamide Adenine Dinucleotide (NADH) under the action of glutamate dehydrogenase (GIDH), namely:
the oxidized NADH amount and the ammonia amount are in a linear relation, and the ammonia content can be estimated by determining the NADH amount through a 340nm absorbance value. In the method, a process of adding reagents in multiple steps exists in the operation process, the reaction time is strictly controlled in each step, the result is greatly influenced by manual operation and is easy to fluctuate, and the ultraviolet absorption value is measured every 2 minutes in the last step until the absorbance is stable, so that the operation is complicated.
Zhang Yongling literature (Zhang Yongling, et al, J Chinese Law medicine for relationship between myocardial amino acid metabolism and early acute myocardial ischemia, 1993, 02) reports that by ligating the anterior descending branch of left coronary artery of rabbit, the degradation of protein and amino acid metabolism during acute myocardial ischemia are studied. When the content of free amino acids (acidic, neutral and basic) and ammonia in ischemic myocardial tissues is measured by ion exchange chromatography, the content of most of the free amino acids is increased during ischemia, the content of alanine, valine, isoleucine and histidine is obviously increased after 15min of ischemia, but the total amount of threonine, glutamic acid and free amino acids is gradually reduced along with the prolongation of the ischemia time. The serine content is increased in the early ischemia stage, and the content is not obviously changed after 60-480 min of ischemia. After 120min of ischemia, the glycine and arginine content gradually decreased. The tissue ammonia content rises early in ischemia and falls late in ischemia. The decrease in total free amino acids, threonine, glycine, arginine and tissue ammonia content is a characteristic change in the metabolism of ischemic myocardial amino acids, and determination of the content of ischemic myocardial free amino acids and tissue ammonia is considered to contribute to the early diagnosis of acute myocardial ischemia. However, the present inventors have found that the aforementioned method of the zhangongliang literature for the detection of free ammonia in biological samples (i.e., myocardial tissue) is not suitable for the detection of free ammonia in commercially available compound amino acid injections.
Therefore, for the quality control department or the drug inspection and detection mechanism of the drug manufacturing enterprise which needs to measure the content of the free ammonia batch by batch in a large scale, the establishment of a rapid, simple, sensitive and stable method for measuring the free ammonia in the compound amino acid injection is urgent.
Disclosure of Invention
The invention aims to provide a qualitative and quantitative determination method for free ammonia in compound amino acid injection, which is expected to have one or more effects of rapidness, simplicity, convenience, sensitivity, stability and the like. It has been found that one or more of the above-mentioned technical effects can be obtained by the method of the present invention, and the present invention has been completed based on this finding.
Therefore, the invention provides a method for determining free ammonia in compound amino acid injection in a first aspect, which comprises the following steps:
(1) providing an assay device: providing an amino acid analyzer and a column (for example, the amino acid analyzer is of Hitachi L-8900 type; for example, the column is a column in which a sodium ion type sulfonic acid group strongly acidic cation exchange resin is used as a packing material, for example, the column has a specification of 4.6 mm. times.60 mm, 3 μm);
(2) preparation of buffers for isolation analysis:
isolation buffers B1, B2, B3, B4 and B5 were prepared according to the following table:
| reagent | B1 | B2 | B3 | B4 | B5 |
| Water (W) | 700ml | 700ml | 700ml | 700ml | 700ml |
| Sodium citrate, measured as its dihydrate | 6.19g | 7.74g | 13.31g | 26.67g | - |
| Sodium hydroxide | 0.24g | 0.8g | - | - | 8.0g |
| Sodium chloride | 5.66g | 7.07g | 3.74g | 54.35g | - |
| Citric acid, measured as the monohydrate thereof | 19.8g | 22.0g | 12.8g | 6.1g | - |
| Ethanol | 135.0ml | 25.0ml | 9.0ml | - | 100.0ml |
| Adding water to constant volume | 1000ml | 1000ml | 1000ml | 1000ml | 1000ml； |
(3) Preparing a derivatization reaction reagent:
filling 979ml of ethylene glycol monomethyl ether into nitrogen for 5 minutes, adding 83mg of sodium borohydride, and continuing to fill nitrogen for 30 minutes to serve as color development liquid R1 for amino acid analysis;
taking 401ml of ethylene glycol monomethyl ether, placing the ethylene glycol monomethyl ether in a 1000ml measuring flask, adding 204g of anhydrous sodium acetate, adding 123ml of glacial acetic acid, adding 336ml of water, dissolving, adding water to a constant volume to scale, and filling nitrogen for 30 minutes to serve as color development liquid R2 for amino acid analysis;
taking 50ml of ethanol, adding water to dilute the ethanol to 1000ml, and taking the ethanol as a color development liquid R3 for amino acid analysis;
(the color-developing solutions R1, R2, and R3 may be referred to as derivatization reagents, derivatization reagents, etc.)
(4) Chromatographic conditions are as follows:
as mobile phase buffer B1, B2, B3, B4 and B5 flow rate is 0.40ml/min, derivatization reagent R1, R2, R3 flow rate is 0.35ml/min, column temperature is 57 ℃, reactor temperature is 135 ℃, detection wavelength is 570nm, sample amount is 5 μ l, gradient elution is carried out according to the following table:
| time in minutes | B1，％ | B2，％ | B3，％ | B4，％ | B5，％ | R1，％ | R2，％ | R3，％ |
| 0.0 | 100 | 0 | 0 | 0 | 0 | 50 | 50 | 0 |
| 3.0 | 100 | 0 | 0 | 0 | 0 | |||
| 3.1 | 0 | 100 | 0 | 0 | 0 | |||
| 6.0 | 0 | 100 | 0 | 0 | 0 | |||
| 6.1 | 0 | 0 | 100 | 0 | 0 | |||
| 14.8 | 0 | 0 | 100 | 0 | 0 | |||
| 14.9 | 0 | 0 | 0 | 100 | 0 | |||
| 29.0 | 0 | 0 | 0 | 100 | 0 | |||
| 29.1 | 0 | 0 | 0 | 0 | 100 | |||
| 32.0 | 50 | 50 | 0 | |||||
| 32.1 | 100 | |||||||
| 33.0 | 0 | 0 | 0 | 0 | 100 | |||
| 33.1 | 0 | 100 | 0 | 0 | 0 | |||
| 34.0 | 0 | 100 | 0 | 0 | 0 | |||
| 34.1 | 100 | 0 | 0 | 0 | 0 | |||
| 37.0 | 0 | 0 | 100 | |||||
| 37.1 | 50 | 50 | 0 | |||||
| 53.0 | 100 | 0 | 0 | 0 | 0 | 50 | 50 | 0 |
(5) Preparing a test solution: precisely measuring a proper amount of compound amino acid injection to be tested, and diluting with ultrapure water (prepared by a pure water machine, and the resistivity is more than or equal to 18.2M omega) until the total amino acid content is 0.8% M/v to be used as a test solution;
(6) preparing a reference substance solution: precisely weighing a proper amount of an ammonium chloride reference substance (which can be easily obtained from the market, for example, from the national pharmaceutical group chemical reagent company, and the purity of which is 100%), adding ultrapure water to dissolve the ammonium chloride reference substance to prepare a reference substance solution containing free ammonia in a concentration range of 0.75-12.5 [ mu ] g/ml (for example, preparing a reference substance solution with approximately similar concentration, for example, a reference substance solution with concentration of 0.75 [ mu ] g/ml, 1 [ mu ] g/ml, 1.5 [ mu ] g/ml, 2 [ mu ] g/ml, 2.5 [ mu ] g/ml, 5 [ mu ] g/ml, 7.5 [ mu ] g/ml, 10 [ mu ] g/ml, 12.5 [ mu ] g/ml and the like according to the concentration of the free ammonia preliminarily measured in the compound amino acid injection to be measured);
(7) and (3) chromatographic determination: respectively and precisely measuring 2-20 mul (such as 2 mul, 5 mul, 10 mul, 20 mul, such as 5 mul) of the test solution and the reference solution, injecting the test solution and the reference solution into a liquid chromatograph, recording a chromatogram, calculating the concentration of free ammonia in the test solution by peak area according to an external standard method, and calculating the content of the free ammonia in the compound amino acid injection according to the preparation method of the test solution.
The method according to the first aspect of the present invention, wherein the amino acid analyzer is Hitachi model L-8900.
The method according to the first aspect of the present invention, wherein the column is a column in which a sodium ion type sulfonic acid group strongly acidic cation exchange resin is a filler.
The method according to the first aspect of the invention, wherein the chromatography column has a size of 4.6mm x 60mm, 3 μm.
The process according to the first aspect of the invention, wherein the amount of sodium citrate added to the buffer is measured as its dihydrate.
The process according to the first aspect of the present invention, wherein the amount of citric acid added to the buffer is measured as the monohydrate.
The method according to the first aspect of the present invention, wherein the ultrapure water used is ultrapure water having a resistivity of 18.2 M.OMEGA.prepared by a pure water machine.
The method according to the first aspect of the invention, wherein the concentration of free ammonia in the control solution is in the range of 0.75-12.5. mu.g/ml, such as in the range of 1-10. mu.g/ml, such as 0.75. mu.g/ml, 1. mu.g/ml, 1.5. mu.g/ml, 2. mu.g/ml, 2.5. mu.g/ml, 5. mu.g/ml, 7.5. mu.g/ml, 10. mu.g/ml, 12.5. mu.g/ml.
The method according to the first aspect of the invention, wherein the chromatography sample volume is 2-20. mu.l, such as 5-15. mu.l, such as 5. mu.l.
Further, the invention provides a compound amino acid injection in the second aspect, which is detected by the method in any embodiment of the first aspect of the invention.
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. 0.05% w/v calcium sodium edetate was also added to buffer B4 in example 1 below, and all test results were obtained with this reagent added. In a supplementary test of the present invention (which may be referred to as supplementary test 1), the buffer solution is not added with the sodium calcium edetate in the above amount, and as a result, the separation degree of the free ammonia peak from the subsequent chromatographic peak in the chromatogram of the test solution is only 1.23, which shows that the separation degree is unacceptable, which greatly affects the accuracy of the method, especially the integral accuracy of the peak area. Thus in one embodiment of any aspect of the invention, 0.05% w/v calcium sodium edetate is also added to buffer B4. In another experiment of the present invention, which may be referred to as example 2, using the methods and conditions of example 1 herein, several batches of commercially available compound amino acid injection (9AA, H20033657), compound amino acid injection (15AA, H19993475), compound amino acid injection (17 AA-i, H44024796), compound amino acid injection (18AA, H24799), compound amino acid injection (18 AA-ix, H20050224), compound amino acid injection (20AA, H20064829)) were tested for free ammonia content, and as a result: the parameters (except for the degree of separation) in the "methodological validation" above are essentially the same as in example 1; the separation of free ammonia from other adjacent peaks was about 2.7 or greater than 4 (3-9 amino acids, possibly with no added component at about 24 min). The attention points of compound amino acid injection and clinical application thereof are described in detail in high-purity literature (high purity, et al, electronic journal of tumor metabolism and nutrition, 2019-02), for example, amino acid is a basic substance of protein required by human nutrition, and is also a precursor of other molecules synthesized by organisms such as antibodies, hormones, enzymes and the like, and participates in a series of metabolic reactions in vivo, 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 (18 AA-II), compound amino acid injection (18 AA-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 (18 AA-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.
Drawings
FIG. 1 is a typical control chromatogram with a free ammonia retention time of 22.6 min.
FIG. 2 is a chromatogram of a sample of the compound amino acid injection, and the separation degree of free ammonia from other adjacent peaks is excellent.
Figure 3 is a spectrum of a blank solvent showing no chromatographic peaks at free ammonia retention time.
Detailed Description
The present application can be further described by the following examples, however, the scope of the present application is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the present application without departing from the spirit and scope of the application. The materials used in the tests and the test methods are generally and/or specifically described herein. Although many materials and methods of operation are known in the art for the purposes of this application, this application is nevertheless described herein in detail as far as possible. The following examples further illustrate the present application without limiting the same.
The following preparation steps are given for the purpose of illustration and are described in some detail based on the comparability of the examples, from which the products or processes referred to in the present application can be fully summarized by the person skilled in the art on the basis of the prior knowledge.
Example 1: detecting free ammonia in compound amino acid injection
1. Instruments and devices:
amino acid analyzer (Hitachi model L-8900); chromatographic column using sodium ion sulfonic acid strong acid cation exchange resin as filler (specification: 4.6 mm. times.60 mm, 3 μm)
2. Preparation of a separation buffer (mobile phase):
buffers B1, B2, B3, B4 and B5 for amino acid isolation were prepared as in table 1 below:
table 1: buffer solution preparation table for separation
| Reagent | B1 | B2 | B3 | B4 | B5 |
| Water (W) | 700ml | 700ml | 700ml | 700ml | 700ml |
| Sodium citrate, measured as its dihydrate | 6.19g | 7.74g | 13.31g | 26.67g | - |
| Sodium hydroxide | 0.24g | 0.8g | - | - | 8.0g |
| Sodium chloride | 5.66g | 7.07g | 3.74g | 54.35g | - |
| Citric acid, measured as the monohydrate thereof | 19.8g | 22.0g | 12.8g | 6.1g | - |
| Ethanol | 135.0ml | 25.0ml | 9.0ml | - | 100.0ml |
| Adding water to constant volume | 1000ml | 1000ml | 1000ml | 1000ml | 1000ml |
3. Preparation of reaction solution (also referred to as color-developing solution, derivatization reagent, etc.):
filling 979ml of ethylene glycol monomethyl ether into nitrogen for 5 minutes, adding 83mg of sodium borohydride, and continuing to fill nitrogen for 30 minutes to serve as color development liquid R1 for amino acid analysis;
taking 401ml of ethylene glycol monomethyl ether, placing the ethylene glycol monomethyl ether in a 1000ml measuring flask, adding 204g of anhydrous sodium acetate, adding 123ml of glacial acetic acid, adding 336ml of water, dissolving, adding water to a constant volume to scale, and filling nitrogen for 30 minutes to serve as color development liquid R2 for amino acid analysis;
50ml of ethanol was taken and diluted with water to 1000ml, which was used as a color developing solution R3 for amino acid analysis.
4. Chromatographic conditions are as follows:
the flow rate of the mobile phase B1-B5 is 0.40ml/min, the flow rate of the derivative reagent R1-R3 is 0.35ml/min, and gradient elution is carried out according to the following table; the column temperature is 57 ℃; the temperature of the reactor is 135 ℃; the detection wavelength is 570 nm; the sample amount is 5 mul; gradient elution according to table 2:
table 2: gradient elution chart
| Time (minutes) | B1(％) | B2(％) | B3(％) | B4(％) | B5(％) | R1(％) | R2(％) | R3(％) |
| 0.0 | 100 | 0 | 0 | 0 | 0 | 50 | 50 | 0 |
| 3.0 | 100 | 0 | 0 | 0 | 0 | |||
| 3.1 | 0 | 100 | 0 | 0 | 0 | |||
| 6.0 | 0 | 100 | 0 | 0 | 0 | |||
| 6.1 | 0 | 0 | 100 | 0 | 0 | |||
| 14.8 | 0 | 0 | 100 | 0 | 0 | |||
| 14.9 | 0 | 0 | 0 | 100 | 0 | |||
| 29.0 | 0 | 0 | 0 | 100 | 0 | |||
| 29.1 | 0 | 0 | 0 | 0 | 100 | |||
| 32.0 | 50 | 50 | 0 | |||||
| 32.1 | 100 | |||||||
| 33.0 | 0 | 0 | 0 | 0 | 100 | |||
| 33.1 | 0 | 100 | 0 | 0 | 0 | |||
| 34.0 | 0 | 100 | 0 | 0 | 0 | |||
| 34.1 | 100 | 0 | 0 | 0 | 0 | |||
| 37.0 | 0 | 0 | 100 | |||||
| 37.1 | 50 | 50 | 0 | |||||
| 53.0 | 100 | 0 | 0 | 0 | 0 | 50 | 50 | 0 |
5. Preparing a test solution:
precisely measuring a proper amount of compound amino acid injection, and diluting with ultrapure water (prepared by a pure water machine, the resistivity is more than or equal to 18.2M omega) to obtain a solution of a test sample, wherein the total amino acid content is about 0.8% (M/v).
6. Preparing a reference substance solution:
taking ammonium chloride reference substance (purchased from chemical reagent of national drug group, purity: 100%), adding ultrapure water to prepare into free ammonia (NH)3) About 5. mu.g/ml of the control solution. If the measured concentration of free ammonia in the test solution is significantly different from this concentration, the solution preparation concentration is adjusted so that the concentration of free ammonia in the control solution is similar to the concentration in the test solution, and generally, a concentration in the range of 0.5 to 20. mu.g/ml, particularly 0.75 to 15. mu.g/ml, particularly 0.75 to 12.5. mu.g/ml is suitable according to the linear test herein.
7. And (3) chromatographic determination:
respectively and precisely measuring 2-20 mul (such as 2 mul, 5 mul, 10 mul and 20 mul, in the example, 5 mul) of the test solution and the reference solution, injecting the measured solution and the reference solution into a liquid chromatograph, recording a chromatogram, calculating the concentration of free ammonia in the test solution by peak area according to an external standard method, and calculating the content of the free ammonia in the compound amino acid injection according to the preparation method of the test solution. The sample detected in the embodiment 1 is compound amino acid injection (18 AA-II, H20066335).
8. And (3) verification of methodology:
8.1 Linear
Preparation of a linear solution: accurately weighing ammonium chloride control substance 42.88mg to 100ml measuring flask, adding water to dilute to scale, shaking, and using as stock solution (containing NH) for linear test3The concentration was 136.25. mu.g/ml). Preparation of Linear solution according to Table 3The solution for linear test was listed. Precisely measuring 1-5 μ l of each solution for linear test, injecting into a liquid chromatograph, and recording chromatogram. Linear regression was performed with the concentration as abscissa and the peak area as ordinate. The data are detailed in Table 4 for ammonia (NH)3) And (5) linear experimental results.
Table 3: preparation of Linear solutions
| Linear solution | Volume of Linear stock solution (ml) | Measuring bottle volume (ml) | Ammonia (NH)3) Concentration (μ g/ml) |
| 1 | 0.25 | 50 | 0.681 |
| 2 | 0.25 | 20 | 1.703 |
| 3 | 0.5 | 20 | 3.406 |
| 4 | 1.0 | 20 | 6.813 |
| 5 | 1.0 | 10 | 13.625 |
Table 4: free ammonia (NH)3) Results of the Linear experiment
| Linear solution | Ammonia (NH)3) Concentration (μ g/ml) | Ammonia (NH)3) |
| 1 | 0.681 | 124207 |
| 2 | 1.703 | 288714 |
| 3 | 3.406 | 703662 |
| 4 | 6.813 | 1409118 |
| 5 | 13.625 | 2807765 |
And (3) test results: ammonia (NH)3) Ammonia (NH) in the range of 0.681-13.625 mug/ml3) The linear equation of the concentration and the peak area is that y is 208805x-28616, and the correlation coefficient r is 0.9997; meets the general test requirements in the field.
8.2 accuracy
Accuracy was examined as recovery, at 6. mu.g/ml ammonia (NH)3) The recovery tests were performed at 80%, 100%, 120% levels to verify the accuracy of the process.
Precisely measuring 2ml of compound amino acid injection (18 AA-II), placing into a 20ml measuring flask, and adding stock solution (NH) for linear test3Content 136.25 mug/ml) 0.4ml, 0.5ml, 0.6ml, (n is 3 × 3), adding water to dilute to scale, shaking up, injecting sample respectively, recording chromatogram, calculating recovery rate according to external standard method. The specific results are shown in Table 5.
Table 5: ammonia (NH)3) Sample adding recovery rate experimental result
And (4) conclusion: ammonia (NH)3) The recovery rate of the method is in the range of 97.80-99.04%, the average recovery rate is 98.1%, the RSD is 0.6%, and the method meets the general test requirements in the field.
8.3 precision
Precision was investigated in a reproducibility test: 6 parts of sample solution with 100% recovery rate in terms of accuracy are prepared to be used as repetitive sample solution, and the measurement is carried out in terms of accuracy method to examine the precision of the method. The results are detailed in Table 6 for ammonia (NH)3) And (5) repeatability experiment results.
Table 6: results of ammonia (NH3) repeat experiments
8.4 quantitation Limit and detection Limit
Ammonia (NH)3) Preparing a quantitative limit solution and a detection limit solution: take solution 4 for linearity test (Ammonia (NH)3) Concentration of 6.813 mug/ml), diluting step by step, injecting into an amino acid analyzer for determination until the S/N ratio is 10:1 as the quantitative limit and the S/N ratio is 3:1 as the detection limit. Measured, ammonia (NH)3) The limit of quantitation was 0.43. mu.g/ml and the limit of detection was 0.14. mu.g/ml.
8.5 solution stability
Take the linear test solution 4 (containing ammonia (NH)3)6.813 μ g/ml), left at room temperature, and ammonia (NH) was measured at 0 hour, 2 hours, 4 hours, and 8 hours, respectively3) In an amount of ammonia (NH)3) Peak area relative standard deviation solution stability was investigated. The results are detailed in Table 7.
Table 7: ammonia (NH)3) Results of stability experiments
As can be seen from the above results, ammonia (NH)3) The reference solution is stable within 8 hours at room temperature and meets the test requirements. Meets the general test requirements in the field.
Typical profile for this example 1: FIG. 1 is a typical control chromatogram with a free ammonia retention time at 22.6 min; FIG. 2 is a chromatogram of a test sample of the compound amino acid injection, wherein the separation degree of free ammonia and other adjacent peaks reaches 2.76, and the general requirement that the separation degree generally reaches more than 1.5 is met; fig. 3 is a spectrum of a blank solvent used to formulate a control solution or a test solution, showing no chromatographic peak at free ammonia retention time.
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 (11)
1. The method for determining free ammonia in the compound amino acid injection comprises the following steps:
(1) providing an assay device: providing an amino acid analyzer and a chromatographic column; the amino acid analyzer is of Hitachi L-8900 type, and the chromatographic column is a chromatographic column taking sodium ion type sulfonic strong acid cation exchange resin as a filler;
(2) preparation of buffers for isolation analysis:
isolation buffers B1, B2, B3, B4 and B5 were prepared according to the following table:
0.05% w/v calcium sodium edetate is also added into the buffer B4;
(3) preparing a derivatization reaction reagent:
filling 979ml of ethylene glycol monomethyl ether into nitrogen for 5 minutes, adding 83mg of sodium borohydride, and continuing to fill nitrogen for 30 minutes to serve as color development liquid R1 for amino acid analysis;
taking 401ml of ethylene glycol monomethyl ether, placing the ethylene glycol monomethyl ether in a 1000ml measuring flask, adding 204g of anhydrous sodium acetate, adding 123ml of glacial acetic acid, adding 336ml of water, dissolving, adding water to a constant volume to scale, and filling nitrogen for 30 minutes to serve as color development liquid R2 for amino acid analysis;
taking 50ml of ethanol, adding water to dilute the ethanol to 1000ml, and taking the ethanol as a color development liquid R3 for amino acid analysis;
(4) chromatographic conditions are as follows:
as mobile phase buffer B1, B2, B3, B4 and B5 flow rate is 0.40ml/min, derivatization reagent R1, R2, R3 flow rate is 0.35ml/min, column temperature is 57 ℃, reactor temperature is 135 ℃, detection wavelength is 570nm, sample amount is 5 μ l, gradient elution is carried out according to the following table:
(5) preparing a test solution: precisely measuring a proper amount of the compound amino acid injection of the test sample, and diluting with ultrapure water until the total amino acid content is 0.8% m/v to obtain a test sample solution;
(6) preparing a reference substance solution: precisely weighing a proper amount of an ammonium chloride reference substance, adding ultrapure water for dissolving to prepare a reference substance solution containing free ammonia with the concentration of 0.75-12.5 mu g/ml;
(7) and (3) chromatographic determination: respectively and precisely measuring 2-20 mul of each of the test solution and the reference solution, injecting the test solution and the reference solution into a liquid chromatograph, recording a chromatogram, calculating the concentration of free ammonia in the test solution according to the peak area by an external standard method, and calculating the content of the free ammonia in the compound amino acid injection according to the preparation method of the test solution.
2. The method according to claim 1, wherein the chromatographic column has a size of 4.6mm x 60mm, 3 μm.
3. The process according to claim 1, wherein the amount of sodium citrate added to the buffer is measured as its dihydrate.
4. The process according to claim 1, wherein the citric acid is added to the buffer in an amount measured as the monohydrate.
5. A process as claimed in claim 1, wherein the ultrapure water used is ultrapure water having a resistivity of 18.2M Ω or more which has been prepared by a water purification machine.
6. The method according to claim 1, wherein the free ammonia concentration in the control solution is in the range of 0.75-12.5 μ g/ml.
7. The method according to claim 1, wherein the free ammonia concentration in the control solution is in the range of 1-10 μ g/ml.
8. The method according to claim 1, wherein the free ammonia concentration in the control solution is 0.75 μ g/ml, 1 μ g/ml, 1.5 μ g/ml, 2 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 7.5 μ g/ml, 10 μ g/ml, or 12.5 μ g/ml.
9. The method according to claim 1, wherein the chromatography sample volume is 2 to 20 μ l.
10. The method according to claim 1, wherein the chromatography sample volume is 5 to 15 μ l.
11. The method according to claim 1, wherein the chromatography sample volume is 5 μ l.
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