Method for detecting cystine and derivatives thereof

Abstract

The invention provides a method for simultaneously detecting at least two of cysteine, acetylcysteine and cystine or salts of amino acids in an amino acid composition. High performance liquid chromatography is adopted for detection, and chromatographic conditions comprise that octadecylsilane chemically bonded silica is adopted as a chromatographic column of a filler, and a mobile phase A and a mobile phase B are adopted for gradient elution; wherein the mobile phase A is a mixed solution of an ammonium sulfate solution and an ion pair reagent. According to the method disclosed by the invention, cysteine, acetylcysteine, cystine or salts of the amino acids in the amino acid composition can be separated from one another and can be effectively separated from other amino acid components or impurities, and the separation degree is high; cysteine, cystine and acetylcysteine in the reference substance and amino acid composition are stable in retention time and good in peak shape. The method provided by the invention has the advantages of strong specificity, good stability, high accuracy, accurate, reliable and effective detection result, good repeatability and high precision.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, in particular to a method for simultaneously detecting at least two of cysteine ​​or its salt, acetylcysteine ​​and cystine in an amino acid composition. Background Art

[0002] Compound amino acid injection or oral solution is an important type of medical and nutritional supplement product, which is mainly used to provide the amino acids needed by the human body and support protein synthesis and metabolism. It is widely used in clinical practice for postoperative recovery, malnutrition, liver disease, kidney disease and other conditions. This type of product usually provides amino acid injection or oral solution with different formulas and concentrations according to different medical needs and specific conditions of patients.

[0003] There are many types of amino acids on the market, such as compound amino acid injection (17AA, 17AA-Ⅱ, 17AA-Ⅲ), compound amino acid injection (18AA-Ⅵ), compound amino acid injection (18AA-Ⅶ), compound amino acid injection (18AA-Ⅸ), compound amino acid injection (20AA for children), etc. These products all contain cysteine. However, cysteine ​​is easily oxidized and degraded, resulting in unstable product quality, which in turn affects the safety and effectiveness of the drug.

[0004] Patent CN115317590A discloses that acetylcysteine ​​can be used as an active ingredient in a compound amino acid injection instead of cysteine. However, in certain oxidation processes or chemical conditions, the acetyl group of acetylcysteine ​​breaks or is removed, releasing cysteine, which is easily oxidized and degraded into cystine. This further exacerbates the instability of the quality of such products.

[0005] Therefore, it is necessary to accurately qualitatively and quantitatively analyze the content of cysteine ​​or acetylcysteine ​​and its common degradation products in compound amino acid injections to meet pharmacopoeia and regulatory requirements. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a method for simultaneously detecting at least two of cysteine, acetylcysteine ​​and cystine or the salts of the above-mentioned amino acids in an amino acid composition. The method of the present invention can separate cysteine, acetylcysteine, cystine or the salts of the above-mentioned amino acids in the amino acid composition from each other, and can effectively separate from other amino acid components or impurities, with high separation; the retention time of cysteine, acetylcysteine, cystine or the salts of the above-mentioned amino acids in the reference substance and the amino acid composition is stable and the peak shape is good. The method of the present invention has strong specificity, good stability and high accuracy, and the detection results are accurate, reliable, effective, reproducible and precise, and can be widely used in the qualitative and quantitative analysis of cysteine, acetylcysteine ​​and cystine substances in amino acid compositions.

[0007] It should be stated that the present invention is based on the inventor's discovery and understanding of the following problems:

[0008] It is common in the art to detect the content of cysteine ​​in an amino acid composition (such as a compound amino acid injection) by high performance liquid chromatography. However, it is very difficult to simultaneously detect two or three cysteines including cysteine ​​and their substitutes and related substances (such as cysteine ​​substitute acetylcysteine ​​and cystine, an oxidative degradation product of cysteine) by this method. This is because the simultaneous detection of cysteine, cystine and acetylcysteine ​​by high performance liquid chromatography has low separation and is difficult to effectively separate. The precision, repeatability and accuracy of the detection cannot meet the requirements of accurate quantification and cannot meet the pharmacopoeia and regulatory requirements.

[0009] Among the above three compounds, acetylcysteine ​​has the highest hydrophobicity due to the introduction of acetyl group, cystine has the second highest hydrophobicity due to the presence of disulfide bond, and cysteine ​​has the lowest hydrophobicity due to the polarity of thiol group. This difference in hydrophobicity can be used to optimize the separation conditions of HPLC. The inventor found through a large number of experiments that when a chromatographic column with octadecylsilane bonded silica gel as filler is used, a mixed solution of ammonium sulfate solution and ion pair reagent is used as mobile phase A, and an organic phase (such as methanol) is used as mobile phase B, when gradient elution is performed, cysteine, cystine, and acetylcysteine ​​have good separation; when the pH value of mobile phase A is further controlled to be 1.5 to 2.5, the separation of these three substances can be further improved to achieve effective separation. The test results show that the mixed solution of ammonium sulfate solution and ion pair reagent is necessary for separating cysteine, cystine, and acetylcysteine ​​from each other, and for effectively separating cystine, cystine, and acetylcysteine ​​from other amino acid components or impurities. Compared with ammonium sulfate solution without ion pair reagent, alkyl sodium sulfonate buffer without ammonium sulfate, ammonium acetate buffer solution containing ion pair reagent, and phosphate buffer containing ion pair reagent, a mixed solution of ammonium sulfate solution and ion pair reagent is used as mobile phase A, and organic compound is used as mobile phase B, and gradient elution is performed, which can achieve effective separation of the above three substances, further optimize retention time, obtain chromatographic peaks with stable retention time and good peak shape, and achieve accurate quantification of cysteine, cystine, and acetylcysteine. The inventor has carried out methodological verification such as specificity, solution stability and accuracy for the above method. The results show that the method has strong specificity, good stability, high accuracy, and accurate, reliable, effective, repeatable and high precision test results.

[0010] Therefore, in the first aspect of the present invention, the present invention proposes a method for simultaneously detecting at least two of cysteine, acetylcysteine ​​and cystine or the salts of the above-mentioned amino acids in an amino acid composition. According to an embodiment of the present invention, high performance liquid chromatography is used for detection, and the chromatographic conditions include: using octadecylsilane bonded silica gel as a chromatographic column as a filler, and using mobile phase A and mobile phase B for gradient elution; wherein the mobile phase A is a mixed solution of ammonium sulfate solution and an ion pair reagent. The method according to an embodiment of the present invention can separate cysteine, acetylcysteine, cystine or the salts of the above-mentioned amino acids in the amino acid composition from each other, and can be effectively separated from other amino acids or impurities, with high separation; the retention time of cysteine, acetylcysteine, cystine or the salts of the above-mentioned amino acids in the reference substance and the amino acid composition is stable and the peak shape is good. The method of the present invention has strong specificity, good stability, high accuracy, accurate, reliable, effective, repeatable and high precision, and can be widely used in the qualitative and quantitative analysis of cysteine, acetylcysteine ​​and cystine in the amino acid composition.

[0011] In high performance liquid chromatography (HPLC), the term "mobile phase B" is often referred to as the organic phase or strong eluent, used together with "mobile phase A (aqueous phase or weak eluent)" to form a gradient elution system.

[0012] In some optional embodiments of the present invention, mobile phase B may include, including but not limited to: methanol, acetonitrile, isopropanol, acetone.

[0013] In the pharmaceutical field, amino acid salts are common compounds that usually have good solubility and stability and are easy to transport and absorb in aqueous solutions. In pharmaceutical preparations, selecting appropriate amino acid salts can improve the bioavailability and efficacy of drugs. In nutritional supplements, amino acid salts can be used as active ingredients to help supplement the amino acids needed by the human body. Exemplarily, the amino acid salts described herein include but are not limited to: hydrochloride, acetate, malate, citrate.

[0014] In some optional embodiments of the present invention, the salt of the amino acid is hydrochloride, acetate, malate or citrate, preferably hydrochloride.

[0015] According to an embodiment of the present invention, based on the volume of the mobile phase A, the final concentration of the ammonium sulfate solution is 0.01-0.1 mol / L, preferably 0.02-0.05 mol / L, and more preferably 0.025-0.045 mol / L. The inventors have determined the preferred concentration of ammonium sulfate solution in mobile phase A through a large number of experiments. The ammonium sulfate solution within this concentration range helps to further improve the separation between cysteine, cystine and acetylcysteine, helps to optimize the retention time, and thus obtains a better chromatographic peak shape. The ammonium sulfate solution within this concentration range also helps to maintain the stability of the high performance liquid chromatography system, reduce background noise, improve the reliability of analysis, help to reduce the interference of other non-detection target components in the amino acid composition, and improve the accuracy and repeatability of detection.

[0016] According to an embodiment of the present invention, the ion pair reagent is a sodium alkyl sulfonate compound. Through the hydrophobic interaction between the hydrophobic alkyl chain of the sodium alkyl sulfonate and the hydrophobic compound, and the sulfonic acid group (-SO 3 - ) and the charge-dipole or ion-dipole interaction between the polar groups of hydrophobic compounds (such as the thiol group -SH of cysteine), and the formation of ion pairs between the sodium alkyl sulfonate compounds and cysteine, cystine, and acetylcysteine ​​further increase the interaction of these three compounds with octadecylsilane bonded silica gel, thereby adjusting their retention time and obtaining better separation and peak shape.

[0017] In some optional embodiments of the present invention, the ion pair reagent includes at least one of sodium octane sulfonate, sodium heptane sulfonate, sodium butane sulfonate, sodium decane sulfonate, sodium tetradecane sulfonate, sodium hexane sulfonate, sodium cyclohexane sulfonate, sodium succinate, and sodium dialkyl sulfonate.

[0018] According to an embodiment of the present invention, the ion pair reagent includes at least one of sodium octane sulfonate and sodium heptane sulfonate. Thus, the separation degree of cysteine, cystine and acetylcysteine ​​is further improved, and the peak shape is improved.

[0019] According to an embodiment of the present invention, based on the volume of the mobile phase A, the final concentration of the ion pair reagent is 0.005-0.06 mol / L, preferably 0.0075-0.035 mol / L, and more preferably 0.010-0.030 mol / L. Thus, the separation effect of the above three compounds in octadecylsilane bonded silica gel high performance liquid chromatography is further improved.

[0020] According to an embodiment of the present invention, the pH value of the mobile phase A mixed solution is 1.5 to 2.5. Thus, the separation degree of the above three compounds in octadecylsilane bonded silica gel high performance liquid chromatography is further improved.

[0021] According to an embodiment of the present invention, the pH value of the mobile phase A is adjusted by acid.

[0022] In some optional embodiments of the present invention, the acid is selected from hydrochloric acid, phosphoric acid, sulfuric acid, oxalic acid, formic acid, oxalic acid, citric acid, and acetic acid. According to the method of the embodiment of the present invention, the pH value of the mobile phase A is precisely controlled to improve the selectivity of the octadecylsilane bonded silica gel chromatographic column.

[0023] According to an embodiment of the present invention, the acid is hydrochloric acid or phosphoric acid, thereby achieving accurate pH value adjustment.

[0024] According to an embodiment of the present invention, the pH value of the mobile phase A is 1.5, 2.0 or 2.5. The inventors have determined the optimal pH value through a large number of experiments. The pH value is easy to achieve precise control to ensure a better separation effect.

[0025] Illustratively, in some optional embodiments of the present invention, the pH value of the mobile phase A can be: 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5.

[0026] According to an embodiment of the present invention, the chromatographic conditions also include: a detection wavelength of 195nm-210mm, an injection volume of 10-70μL, a flow rate of 0.8-1.0mL / min, and a column temperature of 25°C-35°C. Thus, the chromatographic separation effect is further improved, and the precision, accuracy, and repeatability of the detection method are improved.

[0027] According to an embodiment of the present invention, the detection wavelength is 195 nm, 200 nm, 205 nm or 210 nm, preferably 205 nm, thereby further improving the detection sensitivity and the specificity of the analysis.

[0028] According to an embodiment of the present invention, the injection volume is 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, preferably 50 μL. The inventors have determined the optimal sample volume after a large number of experiments. The sample volume can ensure that the detection signal is stable and repeatable, while avoiding the decrease in separation effect caused by overloading of the chromatographic column sample.

[0029] According to an embodiment of the present invention, the flow rate is 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, preferably 0.8 mL / min. Thus, good separation is ensured and detection and analysis are completed within a reasonable time.

[0030] According to an embodiment of the present invention, the column temperature is 25° C., 30° C. or 35° C. The inventors have determined the optimal column temperature after a large number of experiments. The column temperature of the chromatographic strip can reduce the change of chromatographic peaks caused by temperature changes and further improve the chromatographic separation effect.

[0031] According to an embodiment of the present invention, the mobile phase B is methanol.

[0032] In some specific embodiments of the present invention, the gradient elution procedure is as follows:

[0033]

[0034]

[0035] The inventors have determined the above gradient elution procedure after a large number of experiments. Under this elution condition, cystine, acetylcysteine ​​and cysteine ​​in the compound amino acid injection (17AA-II) can be completely separated from other impurities and components.

[0036] According to a specific embodiment of the present invention, the gradient elution procedure is as follows:

[0037] Retention time (min) Mobile phase A (%) Mobile phase B (%) 0 97 3 20 85 15 40 85 15 45 55 45 80 55 45

[0038] Thus, the chromatographic separation effect is further improved, and the precision, accuracy and repeatability of the detection method are improved.

[0039] According to some specific embodiments of the present invention, the method comprises the following steps:

[0040] (1) dissolving the reference substance in a hydrochloric acid solution and diluting with water to a preset concentration to obtain a reference substance solution; wherein the reference substance comprises at least two of cysteine, acetylcysteine ​​and cystine or salts of the above amino acids; in the reference substance solution, the preset concentration of the cysteine ​​or its salt reference substance solution is 60 μg / mL, the concentration of the cystine or its salt reference substance solution is 10 μg / mL, and the preset concentration of the acetylcysteine ​​or its salt reference substance solution is 2 μg / mL;

[0041] (2) mixing equal volumes of the hydrochloric acid solution and the amino acid composition, and then diluting the amino acid composition to 5 times its volume with water to obtain a test solution;

[0042] (3) Detection is performed using the high performance liquid chromatography method.

[0043] According to some preferred embodiments of the present invention, the chromatographic conditions are: using octadecylsilane bonded silica gel as a chromatographic column as a filler, using mobile phase A and mobile phase B to perform gradient elution according to the following procedure:

[0044] Retention time (min) Mobile phase A (%) Mobile phase B (%) 0 97 3 20 85 15 40 85 15 45 55 45 80 55 45

[0045] ; Wherein, the mobile phase A is a mixed solution of ammonium sulfate solution and ion pair reagent, the pH is 1.5-2.5, based on the volume of the mobile phase A, the final concentration of the ammonium sulfate solution is 0.025-0.045 mol / L, the final concentration of the sodium octane sulfonate and / or sodium heptane sulfonate is 0.010-0.030 mol / L; the mobile phase B is methanol; the detection wavelength is 205 mm, the injection volume is 50 μL, the flow rate is 0.8 mL / min, and the column temperature is 25°C-35°C. Thus, the qualitative and quantitative analysis of cysteine, acetylcysteine ​​and cystine substances in the amino acid composition is further achieved.

[0046] According to an embodiment of the present invention, the cysteine ​​salt is hydrochloride, and the pH of the mobile phase A is adjusted by hydrochloric acid or phosphoric acid.

[0047] According to an embodiment of the present invention, the amino acid composition comprises: at least two of cystine, acetylcysteine, cysteine ​​or salts thereof.

[0048] According to an embodiment of the present invention, the amino acid composition includes: lysine, methionine, threonine, leucine, isoleucine, valine, phenylalanine, tryptophan, tyrosine, histidine, arginine, alanine, serine, glycine, aspartic acid, glutamic acid, proline, taurine, ornithine or one or more of the salts of the above amino acids.

[0049] According to an embodiment of the present invention, the amino acid composition is a compound amino acid oral liquid or injection. The method according to an embodiment of the present invention is particularly suitable for compound amino acid oral liquid or injection, and simultaneously performs qualitative and quantitative analysis on cysteine, acetylcysteine ​​and cystine, and the test results are accurate, reliable, reproducible and precise.

[0050] In some optional embodiments of the present invention, the octadecylsilane bonded silica gel includes but is not limited to: Agilent ZORBAX SB-C18, Phenomenex Luna, Hypersil GOLDTM Dim; the specifications of the octadecylsilane bonded silica gel are: 250mm×4.6mm, 3μm, 4.6mm×250mm, 5μm. As known to those skilled in the art, other chromatographic columns with equivalent performance to the above chromatographic columns using octadecylsilane bonded silica gel as filler can also be selected.

[0051] In the second aspect of the present invention, the present invention proposes the use of the aforementioned method in detecting cysteine ​​or its salt and / or acetylcysteine ​​or its salt and / or cystine or its salt in an amino acid composition.

[0052] According to an embodiment of the present invention, the amino acid composition further comprises: one or more of lysine, methionine, threonine, leucine, isoleucine, valine, phenylalanine, tryptophan, tyrosine, histidine, arginine, alanine, serine, glycine, aspartic acid, glutamic acid, proline, taurine, ornithine or the salts of the above amino acids. According to an embodiment of the present invention, the amino acid composition is a compound amino acid oral solution or injection.

[0053] Illustratively, the compound amino acid injection includes but is not limited to: 15AA, 17AA, 17AA-II, 17AA-III, 18AA-VI, 18AA-VII, 18AA-IX, 18AA-X, and 20AA.

[0054] Those skilled in the art will appreciate that the features and advantages described above for the method are also applicable to this application and will not be described in detail herein.

[0055] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0057] Figure 1 The results of high performance liquid chromatography (HPLC) of the test sample in the method of Example 1 of the present invention (compound amino acid 17AA-II injection is used as the test sample); wherein, the peak position (i.e., retention time) of acetylcysteine ​​is 5.821 min, the peak position (i.e., retention time) of cysteine ​​is 21.206 min, and the peak position (i.e., retention time) of cystine is 38.972 min;

[0058] Figure 2 The results of high performance liquid chromatography of the test sample in the method of Example 2 of the present invention (compound amino acid 18AA-VII injection is used as the test sample); wherein, the peak position (i.e., retention time) of acetylcysteine ​​is 5.823 min, the peak position (i.e., retention time) of cysteine ​​is 21.153 min, and the peak position (i.e., retention time) of cystine is 38.947 min;

[0059] Figure 3 The results of HPLC test of the test sample in the method of Example 3 of the present invention (the pediatric compound amino acid 20AA injection is used as the test sample); wherein, the peak position (i.e., retention time) of acetylcysteine ​​is 5.076 min, the peak position (i.e., retention time) of cysteine ​​is 21.063 min, and the peak position (i.e., retention time) of cystine is 38.885 min;

[0060] Figure 4 The results of HPLC test of the test sample in the method of Comparative Example 1 of the present invention (compound amino acid 17AA-II injection is used as the test sample);

[0061] Figure 5 The results of HPLC test of the test sample in the method of Comparative Example 2 of the present invention (compound amino acid 17AA-II injection is used as the test sample);

[0062] Figure 6 The results of HPLC test of the test sample in the method of Comparative Example 3 of the present invention (Pediatric Compound Amino Acid 20AA Injection is used as the test sample);

[0063] Figure 7 The results of HPLC test of the test sample in the method of Comparative Example 4 of the present invention (compound amino acid 18AA-VII injection is used as the test sample);

[0064] Figure 8The results of HPLC detection after the test sample was spiked in the method of Example 1 of the present invention (compound amino acid 18AA-X injection was used as the test sample); wherein, the peak position (i.e., retention time) of acetylcysteine ​​was 5.371 min, the peak position (i.e., retention time) of cysteine ​​was 19.906 min, and the peak position (i.e., retention time) of cystine was 35.806 min;

[0065] The horizontal axis of the above-mentioned high performance liquid chromatography test result graph is time, with the unit of min (minute), and the vertical axis is the response signal intensity of the detector, with the unit of mAu (milliampere). DETAILED DESCRIPTION

[0066] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0067] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0068] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0069] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0070] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0071] In the embodiment of the present invention, the prescription of the compound amino acid injection (17AA-II) is shown in Table 1.

[0072] Table 1: Prescription of compound amino acid injection (17AA-II)

[0073]

[0074] In the examples of the present invention, the prescription of the compound amino acid injection (18AA-X) is shown in Table 2.

[0075] Table 2: Prescription of compound amino acid injection (18AA-X)

[0076]

[0077]

[0078] In the embodiment of the present invention, the prescription of the compound amino acid injection (20AA for children) is shown in Table 3.

[0079] Table 3: Prescription of Compound Amino Acid Injection for Children (20AA for Children)

[0080]

[0081]

[0082] In the examples of the present invention, the prescription of the compound amino acid injection (18AA-VII) is shown in Table 4.

[0083] Table 4: Prescription of compound amino acid injection (18AA-VII)

[0084]

[0085] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0086] Example 1

[0087] In this example, the compound amino acid solution (17AA-II) was used as a test sample, and cystine, acetylcysteine ​​and cysteine ​​were simultaneously detected by high performance liquid chromatography. The specific method is as follows:

[0088] Octadecylsilane bonded silica gel was used as the filler (Agilent ZORBAX SB-C18, 250 mm × 4.6 mm, 3 μm); ammonium sulfate buffer solution (3.3 g of ammonium sulfate (equivalent to 0.025 mol / L) and 4.3 g of sodium octane sulfonate (0.020 mol / L), dissolved in 1000 mL of water, and the pH value was accurately adjusted to 1.5 with hydrochloric acid solution) was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed according to Table 5; the detection wavelength was 195 nm; the flow rate was 0.8 mL per minute; the column temperature was 25°C; and the injection volume was 50 μL.

[0089] Table 5: Gradient elution program of Example 1

[0090] Retention time (min) Mobile phase A (%) Mobile phase B (%) 0 97 3 30 85 15 50 85 15 55 55 45 90 55 45 95 97 3 135 97 3

[0091] The experimental results are as follows Figure 1 shown.

[0092] The results showed that in the test compound amino acid injection (17AA-II), cystine, acetylcysteine ​​and cysteine ​​could be completely separated from other impurities and components by the HPLC method of Example 1.

[0093] Example 2

[0094] In this example, the compound amino acid injection (18AA-VII) was used as a test sample, and cystine, acetylcysteine ​​and cysteine ​​were simultaneously detected by high performance liquid chromatography. The specific method is as follows:

[0095] Octadecylsilane bonded silica gel was used as the filler (Agilent ZORBAX SB-C18, 250mm×4.6mm, 3μm); ammonium sulfate buffer solution (5.0g of ammonium sulfate (equivalent to 0.038mol / L) and 6.0g of sodium heptane sulfonate (equivalent to 0.030mol / L), dissolved in 1000mL of water, and the pH value was accurately adjusted to 2.0 with hydrochloric acid solution) was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed according to Table 6; the detection wavelength was 210nm; the flow rate was 0.8mL per minute; the column temperature was 35℃; and the injection volume was 50μL.

[0096] Table 6: Gradient elution program of Example 2

[0097] Retention time (min) Mobile phase A (%) Mobile phase B (%) 0 98 2 20 85 15 40 85 15 45 55 45 80 55 45 85 97 3 120 97 3

[0098] The experimental results are as follows Figure 2 shown.

[0099] The results showed that in the test compound amino acid injection (18AA-VII), cystine, acetylcysteine ​​and cysteine ​​could be completely separated from other impurities and components by the HPLC method of Example 2.

[0100] Example 3

[0101] In this example, compound amino acid injection (20AA for children) was used as a test sample, and cystine, acetylcysteine ​​and cysteine ​​were simultaneously detected by high performance liquid chromatography. The specific method is as follows:

[0102] Octadecylsilane bonded silica gel was used as the filler (Phenomenex Luna, 250 mm × 4.6 mm, 3 μm, or a chromatographic column with equivalent performance); ammonium sulfate buffer solution (take 6.0 g of ammonium sulfate (equivalent to 0.045 mol / L) and 2.0 g of sodium heptane sulfonate (equivalent to 0.010 mol / L), add 1000 mL of water to dissolve, and accurately adjust the pH value to 2.5 with hydrochloric acid solution) was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed according to Table 7; the detection wavelength was 205 nm; the flow rate was 0.8 mL per minute; the column temperature was 30°C; and the injection volume was 50 μL.

[0103] Table 7: Gradient elution program of Example 3

[0104] Retention time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 20 75 25 40 75 25 45 60 40 80 60 40 85 97 3 120 97 3

[0105] The experimental results are as follows Figure 3 shown.

[0106] The results showed that in the test compound amino acid injection (20AA for children), cystine, acetylcysteine ​​and cysteine ​​could be completely separated from other impurities and components by the HPLC method of Example 3.

[0107] Comparative Example 1: Mobile phase A uses phosphate buffer containing ion pair reagent

[0108] CN117269359A discloses a method for detecting the content of cysteine ​​in a compound amino acid injection. The inventors refer to the preferred embodiment 8 of CN117269359A and simultaneously detect cystine, acetylcysteine ​​and cysteine ​​in a compound amino acid injection (17AA-II) (test sample) for experiments. The specific test scheme is as follows:

[0109] Octadecylsilane bonded silica gel was used as filler (Hypersil GOLDTM Dim 4.6mm×250mm, 5μm); phosphate buffer (containing 0.02mol / L potassium dihydrogen phosphate, 0.01mol / L sodium heptane sulfonate, pH adjusted to 2.3 with phosphoric acid) was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to Table 8; flow rate was 1.0mL per minute; column temperature was 10℃; gradient elution was performed according to Table 8; detection wavelength was 200nm; injection volume was 10μl. The sample was diluted 5 times with water as the test solution, and an appropriate amount of the reference substance was diluted with water to make a solution with a concentration equivalent to that of the test substance.

[0110] Table 8: Gradient elution program of Comparative Example 1

[0111]

[0112]

[0113] The experimental results are as follows Figure 4 shown.

[0114] The results showed that in the test compound amino acid injection (17AA-II), the components of cystine, acetylcysteine ​​and cysteine ​​could not be completely separated by the HPLC method of Comparative Example 1.

[0115] Comparative Example 2: Mobile phase contains ion pair reagent

[0116] CN117147733A discloses a quantitative detection method for related substances of cysteine ​​raw materials. The inventors refer to Example 1 of CN117147733A and simultaneously detect cystine, acetylcysteine ​​and cysteine ​​in compound amino acid injection (17AA-II) (test sample) for experiments. The specific test scheme is as follows:

[0117] Octadecylsilane bonded silica gel was used as the filler (Shiseido MGⅡC18, 4.6mm×250mm, 5μm); the mobile phase was: 4mmol / L sodium octane sulfonate buffer (0.9g of sodium octane sulfonate was taken, 1000mL of water was added to dissolve it, and the pH value was adjusted to 2.0 with phosphoric acid): acetonitrile = 95:5; column temperature: 30℃; flow rate: 1.0mL / min; wavelength: 200nm; injection volume: 20μl; elution conditions were as follows: isocratic elution.

[0118] The experimental results are as follows Figure 5 shown.

[0119] The results showed that in the test compound amino acid injection (17AA-II), the components of cystine, acetylcysteine ​​and cysteine ​​could not be completely separated by the HPLC method of Comparative Example 2.

[0120] Comparative Example 3: Mobile phase A uses ammonium acetate buffer solution containing ion pair reagent

[0121] Compared with Example 2, the main difference between this comparative example and Example 2 is that the mobile phase A uses an ammonium acetate buffer solution containing an ion pair reagent; at the same time, the test sample is a compound amino acid injection (pediatric 20AA). The specific test plan is as follows:

[0122] Octadecylsilane bonded silica gel was used as filler (Agilent ZORBAX SB-C18, 250 mm × 4.6 mm, 3 μm, or a chromatographic column with equivalent performance); ammonium acetate buffer solution (2.9 g of ammonium acetate and 6.0 g of sodium heptane sulfonate were dissolved in 1000 mL of water, and the pH value was accurately adjusted to 2.0 with hydrochloric acid solution) was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed according to Table 9; the detection wavelength was 205 nm; the flow rate was 0.8 mL per minute; the column temperature was 30°C; and the injection volume was 50 μL.

[0123] Table 9: Gradient elution program of Comparative Example 3

[0124]

[0125]

[0126] The experimental results are as follows Figure 6 shown.

[0127] The results showed that in the test compound amino acid injection (Pediatric 20AA), cystine, acetylcysteine ​​and cysteine ​​could not be completely separated from other impurities and components by the HPLC method of Comparative Example 3.

[0128] Comparative Example 4: Mobile phase A uses ammonium sulfate buffer without ion pair reagent

[0129] Compared with Example 2, the main difference between this comparative example and Example 2 is that the mobile phase A uses an ammonium sulfate buffer solution without an ion pair reagent; and the test sample is a compound amino acid injection (18AA-VII). The specific test plan is as follows:

[0130] Octadecylsilane bonded silica gel was used as filler (Agilent ZORBAX SB-C18, 250 mm × 4.6 mm, 3 μm, or a chromatographic column with equivalent performance); ammonium sulfate buffer solution (5 g of ammonium sulfate was dissolved in 1000 mL of water, and the pH value was accurately adjusted to 2.0 with hydrochloric acid solution) was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed according to Table 10; the detection wavelength was 205 nm; the flow rate was 0.8 mL per minute; the column temperature was 30°C; and the injection volume was 50 μL.

[0131] Table 10: Gradient elution program of Comparative Example 4

[0132] Retention time (min) Mobile phase A (%) Mobile phase B (%) 0 97 3 20 85 15 40 85 15 45 55 45 80 55 45 85 97 3 130 97 3

[0133] The experimental results are as follows Figure 7 shown.

[0134] The results showed that in the test compound amino acid injection (18AA-VII), cystine, acetylcysteine ​​and cysteine ​​could not be completely separated from other impurities and components by the HPLC method of Comparative Example 4.

[0135] Furthermore, the inventors conducted methodological verification of the detection method of Example 1 of the present invention in terms of specificity, solution stability, accuracy, etc.

[0136] Exclusive

[0137] Specificity is the ability of an analytical method to distinguish and accurately measure a target compound, especially in the presence of other potentially similar compounds or impurities.

[0138] The blank sample does not interfere with the determination of acetylcysteine, cystine, and cysteine; the retention time of each component peak in the spiked test solution is basically consistent with the retention time of the main peak of the corresponding reference solution; the separation between the peaks of each component in the spiked test solution is not less than 1.5.

[0139] Among them, the "blank sample" is a solution without any test substance added, and its measurement results will not interfere with the detection of acetylcysteine, cystine and cysteine; the "spike test solution" is a test solution with a known cysteine ​​concentration in which a certain amount of cystine, acetylcysteine ​​and cysteine ​​standards are added.

[0140] 2. Specificity verification results

[0141] The test results of compound amino acid injection (18AA-X) are shown in Table 11. Figure 8 shown.

[0142] Table 11: Specificity verification results of cysteine ​​and its related substances

[0143]

[0144] Note: “ND” means not detected.

[0145] The results show that in the spiked test solution, the separation between cystine, acetylcysteine, cysteine ​​and adjacent peaks is greater than 1.5, and the separation effect is good. This result shows that the chromatographic conditions of the present invention can effectively separate cystine, acetylcysteine, cysteine ​​and other unknown impurities, accurately analyze and detect the content of cystine, acetylcysteine, cysteine ​​and other unknown impurities, and realize the simultaneous detection of cysteine ​​and its related substances in the amino acid composition.

[0146] The above results show that the method of the present invention has strong specificity, good discrimination ability, and accurate and reliable detection results.

[0147] Solution stability

[0148] Solution stability is an important part of analytical methodology validation and is related to the reliability and effectiveness of the analytical method. The purpose of the solution stability test is to ensure that the sample solution and the standard solution remain stable for a certain period of time under the specified storage conditions.

[0149] 1. Acceptance criteria:

[0150] The test was carried out under the planned conditions at a controlled temperature (2-8°C).

[0151] Reference solution: The peak area RSD should be ≤2.0%. After a new sequence is created, the recovery rate of the response factor of the reference solution relative to the response factor of the newly prepared reference solution should be between 98.0% and 102.0%.

[0152] Test solution: Peak area RSD should be ≤2.0%.

[0153] 2. Solution stability verification results

[0154] The results are shown in Tables 12 to 14.

[0155] Table 12: Solution Stability of Acetylcysteine ​​and Cystine Reference Substances

[0156]

[0157]

[0158] Table 13: Solution stability of cysteine ​​hydrochloride reference

[0159]

[0160] Table 14: Solution stability of the test products

[0161]

[0162] The results showed that: (1) under temperature control (2-8°C), the reference solution acetylcysteine ​​and cystine were stable within 172 hours, cysteine ​​was stable within 141.5 hours, and the test solution was stable within 48 hours. (2) under temperature control (2-8°C), the reference solution: acetylcysteine ​​and cystine within 172 hours, the recovery rate of the reference solution response factor was 101.6% and 99.8%, respectively, and the recovery rate of the reference solution response factor was 101.5% within 141.5 hours of cysteine, both of which were between 98.0% and 102.0%; the test solution: within 48 hours, the peak area RSD was 1.7% (≤2.0%).

[0163] The above results show that the solution of the method of the present invention is stable and the detection results are reliable and effective.

[0164] Accuracy

[0165] Accuracy is one of the key indicators for evaluating the performance of analytical methods and is usually evaluated through recovery experiments.

[0166] 1. Acceptance criteria

[0167] The recovery rate of each concentration level calculated by the external standard method should be between 90% and 110%, and the recovery rate RSD should be ≤5.0%.

[0168] 2. Accuracy verification results

[0169] The statistical results are shown in Table 15.

[0170] Table 15: Accurate results of cystine and acetylcysteine

[0171]

[0172]

[0173] The results showed that the recovery rates of cystine at each concentration level calculated by the external standard method of the patented method were between 98.8% and 102.6%, and those of acetylcysteine ​​were between 95.4% and 101.9%. The average recovery rates of cystine and acetylcysteine ​​of 9 samples were 100.9% (RSD 1.5%) and 98.1% (RSD 2.1%).

[0174] The above results show that the method of the present invention has good accuracy, high precision and high consistency of the detection results.

[0175] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for simultaneously detecting at least two of cysteine, acetylcysteine ​​and cystine or salts of the above amino acids in an amino acid composition, characterized in that: The detection was performed by high performance liquid chromatography, and the chromatographic conditions included: using octadecylsilane bonded silica gel as a chromatographic column as a filler, and using mobile phase A and mobile phase B for gradient elution; Wherein, the mobile phase A is a mixed solution of ammonium sulfate solution and ion pair reagent.

2. The method according to claim 1, characterized in that The salt of the amino acid is hydrochloride, acetate, malate or citrate, preferably hydrochloride; optionally, based on the volume of the mobile phase A, the final concentration of the ammonium sulfate solution is 0.01-0.1 mol / L, preferably 0.02-0.05 mol / L, more preferably 0.025-0.045 mol / L.

3. The method according to claim 1, characterized in that The ion pair reagent is a sodium alkyl sulfonate compound; Optionally, the ion pair reagent includes at least one of sodium octane sulfonate, sodium heptane sulfonate, sodium butane sulfonate, sodium decane sulfonate, sodium tetradecane sulfonate, sodium hexane sulfonate, sodium cyclohexane sulfonate, sodium succinate, and sodium dialkyl sulfonate; Optionally, the ion pair reagent includes: at least one of sodium octane sulfonate and sodium heptane sulfonate; Optionally, based on the volume of the mobile phase A, the final concentration of the ion pair reagent is 0.005 to 0.06 mol / L, preferably 0.0075 to 0.035 mol / L, and more preferably 0.010 to 0.030 mol / L.

4. The method according to claim 1, characterized in that: The pH value of the mobile phase A mixed solution is 1.5 to 2.5; Optionally, the pH value of the mobile phase A is adjusted by acid; Further, the acid is selected from hydrochloric acid, phosphoric acid, sulfuric acid, oxalic acid, formic acid, oxalic acid, citric acid, and acetic acid; Optionally, the acid is hydrochloric acid or phosphoric acid; Optionally, the pH value of the mobile phase A is 1.5, 2.0 or 2.

5.

5. The method according to claim 1, characterized in that The chromatographic conditions also include: a detection wavelength of 195nm-210mm, an injection volume of 10-70μL, a flow rate of 0.8-1.0mL / min, and a column temperature of 25°C-35°C; Optionally, the detection wavelength is 195nm, 200nm, 205nm or 210nm, preferably 205nm; Optionally, the injection volume is 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, preferably 50 μL; Optionally, the flow rate is 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, preferably 0.8 mL / min; Optionally, the column temperature is 25°C, 30°C or 35°C.

6. The method according to claim 1, characterized in that The mobile phase B is methanol; Optionally, the gradient elution program is as follows: ， Furthermore, the gradient elution procedure is as follows: 。 7. The method according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (1) dissolving the reference substance in a hydrochloric acid solution and diluting with water to a preset concentration to obtain a reference substance solution; wherein the reference substance comprises at least two of cysteine, acetylcysteine ​​and cystine or salts of the above amino acids; in the reference substance solution, the preset concentration of the cysteine ​​or its salt reference substance solution is 60 μg / mL, the preset concentration of the cystine or its salt reference substance solution is 10 μg / mL, and the preset concentration of the acetylcysteine ​​or its salt reference substance solution is 2 μg / mL; (2) mixing equal volumes of the hydrochloric acid solution and the amino acid composition, and then diluting the amino acid composition to 5 times its volume with water to obtain a test solution; (3) using the high performance liquid chromatography method for detection; Optionally, the chromatographic conditions are: using a chromatographic column with octadecylsilane bonded silica gel as a filler, using mobile phase A and mobile phase B to perform gradient elution according to the following procedure: Wherein, the mobile phase A is a mixed solution of ammonium sulfate solution and an ion pair reagent, the pH is 1.5-2.5, based on the volume of the mobile phase A, the final concentration of the ammonium sulfate solution is 0.025-0.045 mol / L, and the final concentration of the sodium octane sulfonate and / or sodium heptane sulfonate is 0.010-0.030 mol / L; the mobile phase B is methanol; The detection wavelength was 205 mm, the injection volume was 50 μL, the flow rate was 0.8 mL / min, and the column temperature was 25°C to 35°C; Optionally, the cysteine ​​salt is hydrochloride, and the pH of the mobile phase A is adjusted by hydrochloric acid or phosphoric acid.

8. The method according to claim 1, characterized in that The amino acid composition comprises: at least two of cystine, acetylcysteine, cysteine ​​or salts thereof; Optionally, the amino acid composition further comprises: one or more of lysine, methionine, threonine, leucine, isoleucine, valine, phenylalanine, tryptophan, tyrosine, histidine, arginine, alanine, serine, glycine, aspartic acid, glutamic acid, proline, taurine, ornithine or salts thereof; Optionally, the salt of the amino acid is hydrochloride, acetate, malate or citrate, preferably hydrochloride; Optionally, the amino acid composition is a compound amino acid oral solution or injection solution.

9. Use of the method according to any one of claims 1 to 8 in detecting cysteine ​​and / or acetylcysteine ​​and / or cystine or salts of the above amino acids in an amino acid composition.

10. The use according to claim 9, characterized in that: The amino acid composition comprises: at least two of cystine, acetylcysteine ​​or cysteine ​​or the above amino acid salts; Optionally, the amino acid composition further comprises: one or more of lysine, methionine, threonine, leucine, isoleucine, valine, phenylalanine, tryptophan, tyrosine, histidine, arginine, alanine, serine, glycine, aspartic acid, glutamic acid, proline, taurine, ornithine or salts thereof; Optionally, the salt of the amino acid is hydrochloride, acetate, malate or citrate, preferably hydrochloride; Optionally, the amino acid composition is a compound amino acid oral solution or injection solution.

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