Method for detecting impurities in compound amino acid injection

Abstract

The invention provides a method for detecting impurities in a compound amino acid injection, and belongs to the field of pharmaceutical analysis. A high performance liquid chromatography method is adopted, octadecylsilane chemically bonded silica is used as a chromatographic column, a phosphoric acid solution with the concentration of 20.0-25.0 g / L is used as a mobile phase A, a methanol solution is used as a mobile phase B, and gradient elution is performed on the compound amino acid injection so as to detect methionine glycine and / or phenylethylamine and / or benzoic acid and / or phenylpropionic acid and other impurities. The method provided by the invention has high separation degree on impurities introduced or generated by glycine and phenylalanine, can effectively control the content of methionine glycine and / or phenylethylamine and / or benzoic acid and / or phenylpropionic acid in the compound amino acid injection, and improves the safety of the injection; meanwhile, conventional reagents are used in the detection process, the method is environmentally friendly, the detection method is simple, the cost is low, and the method is suitable for practical application and popularization and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a method for detecting impurities in a compound amino acid injection, and belongs to the field of drug analysis. Background Art

[0002] The compound amino acid injection (18AA-VII) involves 18 kinds of raw materials with complex ingredients. At the same time, the compound amino acid injection is widely used in clinical nutritional support. Therefore, it is an important task to strictly control the quality of the injection to ensure the safety of the medication. In order to ensure the safety of the injection, it is necessary to study and detect the impurities that may be introduced in the raw materials. Some existing literature discloses methods for controlling the impurities and degradation products introduced by methionine in the compound amino acid injection, such as the Chinese patent with application number 201610764016.9. This method is suitable for detecting the content of methionine sulfoxide, a degradation product of methionine in amino acid injection, and has strong specificity. At present, there is no relevant detection method for the impurities that may be introduced or generated by glycine and phenylalanine in the compound amino acid injection, and it is difficult to ensure the safety of the amino acid injection.

[0003] In view of this, it is necessary to propose a method for detecting impurities in compound amino acid injection to solve the above problems. Summary of the invention

[0004] The object of the present invention is to provide a method for detecting impurities in a compound amino acid injection, so as to accurately detect impurities that may be introduced or generated by glycine and phenylalanine in the compound amino acid injection.

[0005] To achieve the above object, the present invention provides a method for detecting impurities in a compound amino acid injection, wherein the detection method adopts high performance liquid chromatography, using octadecylsilane bonded silica gel as a chromatographic column, using a phosphoric acid solution with a concentration of 20.0 to 25.0 g / L as a mobile phase A, using methanol as a mobile phase B, and the gradient elution procedure is as follows:

[0006] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95～100 5～0 10 95～100 5～0 25 75～85 25～15 45 35～45 65～55 46 95～100 5～0 65 95～100 5～0

[0007] The impurities include methionylglycine and / or phenethylamine and / or benzoic acid and / or phenylpropionic acid.

[0008] Further, the gradient elution procedure is preferably:

[0009] Time (min) Mobile phase A (%) Mobile phase B (%) 0 98～100 2～0 10 98～100 2～0 25 80～85 20～15 45 38～42 62～58 46 98～100 2～0 65 98～100 2～0 .

[0010] The gradient elution program is more preferably:

[0011] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 25 82 18 45 40 60 46 100 0 65 100 0 .

[0012] Further, the mobile phase A is a phosphoric acid solution with a concentration of 22.0 to 24.0 g / L, preferably, the mobile phase A is a phosphoric acid solution with a concentration of 22.5 to 23.5 g / L, and more preferably, the mobile phase A has a concentration of 23 g / L.

[0013] Furthermore, the chromatographic column is a chromatographic column with performance equivalent to Welch Ultimate LPAQ C18. Preferably, the chromatographic column is Welch Ultimate LPAQ C18, 4.6 mm×250 mm, and the particle size of the silica gel particles is 5 μm.

[0014] Furthermore, the column temperature of the chromatographic column is 25-35°C, preferably, the column temperature is 30°C.

[0015] Furthermore, the flow rate of the mobile phase is 0.5 to 1.5 ml / min, preferably, the flow rate of the mobile phase is 0.9 to 1.0 ml / min, and more preferably, the flow rate of the mobile phase is 1.0 ml / min.

[0016] Further, the injection volume is 3-20 μl, preferably, the injection volume is 5-10 μl, and more preferably, the injection volume is 10 μl.

[0017] Furthermore, the injection temperature is 10-25°C, preferably, the injection temperature is 15°C.

[0018] Furthermore, the detection wavelength of the chromatogram is 200-220 nm, preferably, the detection wavelength is 205-210 nm, and more preferably, the detection wavelength is 205 nm.

[0019] Furthermore, the impurities include methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid.

[0020] Furthermore, the compound amino acid injection includes glycine and / or phenylalanine. Preferably, the compound amino acid injection includes glycine and phenylalanine.

[0021] Furthermore, the model of the compound amino acid injection is 18AA-VII.

[0022] As a further improvement of the present invention, the detection conditions are as follows: the model specification of the chromatographic column is Ultimate LP-AQ4.6mm×250mm, 5μm, the column temperature is 30°C; the mobile phase A is a phosphoric acid solution with a concentration of 23.0g / L, the mobile phase B is methanol, the injection volume is 10μl, the flow rate is 1.0ml / min, and the detection wavelength is 205nm; the gradient elution program is as follows:

[0023] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 25 82 18 45 40 60 46 100 0 65 100 0 .

[0024] The present invention adopts the detection method, which not only solves the above problems well, but also enables the methionylglycine peak, the phenethylamine peak, the benzoic acid peak and the phenylpropionic acid peak to be effectively separated from other components, and all components can be completely eluted without residue, and at the same time, there will be no drift and peak width difference between the impurity peaks in the reference solution and the test solution; in addition, the detection method of the present invention enables the methionylglycine peak, the phenethylamine peak, the benzoic acid peak and the phenylpropionic acid peak to have high separation, better peak shape, less external interference, consistent retention time of the impurity peaks in the reference solution and the test solution, high detection precision and accuracy, and good repeatability.

[0025] The beneficial effects of the present invention are as follows: the method provided by the present invention has a high degree of separation for impurities introduced or generated by glycine and phenylalanine, can effectively detect and control the content of methionylglycine and / or phenylethylamine and / or benzoic acid and / or phenylpropionic acid in the compound amino acid injection, and improves the safety of the injection; at the same time, conventional reagents are used in the detection process, which is environmentally friendly, the detection method is simple and low in cost, is suitable for practical application and promotion, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a partial chromatogram of various solutions in Example 1.

[0027] Figure 2 It is a partial chromatogram of the mixed reference solution, test solution and spiked test solution in Example 2.

[0028] Figure 3 It is a partial chromatogram of the blank solution, mixed reference solution, and spiked test solution in Example 3.

[0029] Figure 4 This is a partial chromatogram of the spiked test solution in Example 4.

[0030] Figure 5 This is a partial chromatogram of the spiked test solution in Example 5.

[0031] Figure 6 This is a partial chromatogram of the spiked test solution in Example 6.

[0032] Figure 7 1 is a partial chromatogram of the spiked test solution in Example 7.

[0033] Figure 8 This is a partial chromatogram of the system suitability experiment in Experimental Example 1.

[0034] Fig. 9 This is a partial chromatogram of the specificity experiment in Experimental Example 2.

[0035] Fig.10 It is the linear graph of the linear experiment in Experimental Example 4. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0037] For ease of description and understanding, the impurities in this application are described as methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid, indicating that the detection method in this application can have a better detection and separation effect on the above four impurities. In other words, when the impurity is only any one or several of the above four impurities, it is also applicable to this detection method.

[0038] In the present invention, the configuration methods of various solutions are as follows:

[0039] Blank solution: water.

[0040] Negative blank sample: Prepare according to the concentration of each raw material and excipient in the following prescription (without adding methionine, glycine and phenylalanine).

[0041] The prescription of 200ml compound amino acid injection is: isoleucine: 1.82g; leucine: 2.58g; lysine acetate: 2.00g; methionine: 0.88g; phenylalanine: 1.40g; threonine: 1.50g; tryptophan: 0.26g; valine: 2.80g; alanine: 1.42g; arginine: 1.80g; aspartic acid: 0.20g; cysteine: 0.07g; glutamic acid: 0.10g; histidine: 1.00g; proline: 1.00g; serine: 0.34g; tyrosine: 0.08g; glycine: 1.40g; sodium bisulfite: 0.06g.

[0042] Negative blank solution: Take 2 ml of negative blank sample and place it in a 10 ml volumetric flask, add water to dilute to the scale, and shake well.

[0043] Methionylglycine reference substance stock solution (200 μg / ml): Take about 10 mg of methionylglycine reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0044] Phenylethylamine reference substance stock solution (200 μg / ml): Take about 13 mg of phenylethylamine hydrochloride reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0045] Benzoic acid reference substance stock solution (200 μg / ml): Take about 10 mg of benzoic acid reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0046] Phenylpropionic acid reference substance stock solution (200 μg / ml): Take about 10 mg of phenylpropionic acid reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0047] Mixed impurity stock solution (20 μg / ml): Accurately measure 5 ml each of methionylglycine reference stock solution, phenylethylamine reference stock solution, benzoic acid reference stock solution and phenylpropionic acid reference stock solution, place them in the same 50 ml volumetric flask, dilute to the scale with water, and shake well.

[0048] Reference solution (2 μg / ml): Accurately measure 1 ml of the mixed impurity stock solution and place it in a 10 ml volumetric flask, dilute to the mark with water, and shake well.

[0049] Test solution: Accurately measure 2 ml of compound amino acid injection (18AA-VII) into a 10 ml volumetric flask, dilute to the mark with water, and shake well.

[0050] Spiked test solution: Accurately measure 2 ml of the test solution and 1 ml of the mixed impurity stock solution, place them in the same 10 ml volumetric flask, dilute to the scale with water, and shake well.

[0051] Example 1

[0052] Chromatographic column: A chromatographic column with octadecylsilane bonded silica as filler, model specification: Ultimate LP-AQ4.6mm×250mm, 5μm.

[0053] Mobile phase: Mobile phase A is methanol-phosphoric acid aqueous solution (volume ratio is 0.5:99.5), and the phosphoric acid aqueous solution contains 2.875g of phosphoric acid in 1L. Mobile phase B is methanol-phosphoric acid aqueous solution (volume ratio is 47.5:52.5), and the phosphoric acid aqueous solution contains 5.477g of phosphoric acid in 1L.

[0054] The column temperature was 30°C, the flow rate was 1.0 ml / min, the detection wavelength was 205 nm, the injection volume was 10 μl, and the blank solvent, phenylpropionic acid reference stock solution, phenylethylamine reference stock solution, benzoic acid reference stock solution, carbazylglycine reference stock solution, test solution and 5-fold diluted test solution were injected and tested respectively.

[0055] Gradient elution program:

[0056] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 6 100 0 50 52 48 60 52 48 61 100 0 75 100 0

[0057] Detection spectrum see Figure 1As shown, under this chromatographic system, the peak positions of methionylglycine and phenylethylamine interfere with the test solution.

[0058] Example 2

[0059] The difference between this embodiment and embodiment 1 is that: a phosphoric acid aqueous solution with a concentration of 23.0 g / l is used as mobile phase A, methanol is used as mobile phase B, the injection volume is 5 μl, and the mixed reference solution, the test solution and the spiked test solution are injected and tested respectively, and the gradient elution program is:

[0060]

[0061]

[0062] Some of the detection spectra can be seen Figure 2 As shown, there is no interference between the elution positions of phenethylamine and other components, and the retention time of the phenethylamine peak in the spiked test solution is consistent with the retention time of the phenethylamine peak in the reference substance, but the separation degree between phenethylamine and the adjacent interfering peak is 1.08, which is relatively small.

[0063] Example 3

[0064] The difference between this embodiment and embodiment 2 is that the blank solution, the mixed reference solution (phenylethylamine, benzoic acid, phenylpropionic acid), the methionylglycine reference solution, the spiked test solution (phenylethylamine, benzoic acid, phenylpropionic acid), and the spiked test solution (methionylglycine) are respectively injected and tested, and the gradient elution program is:

[0065] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 25 82 18 45 40 60 46 100 0 65 100 0

[0066] See the detection spectrum Figure 3 As shown, Figure 3 This is the detection spectrum when the injection volume is 5μl. It can be seen from the figure that the retention time of methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid is consistent, and there is no interference in the peak position of each impurity, but the response of some impurity peaks is small. When the injection volume is increased to 10μl, the response of each impurity peak increases significantly, and the separation degree with adjacent peaks is good.

[0067] Example 4

[0068] Chromatographic column: A chromatographic column with octadecylsilane bonded silica as filler, model specification: Ultimate LP-AQ4.6mm×250mm, 5μm.

[0069] Mobile phase: Aqueous phosphoric acid solution with a concentration of 23.0 g / l was used as mobile phase A, and methanol was used as mobile phase B.

[0070] The column temperature was 30°C, the flow rate was 1.0 ml / min, the detection wavelength was 205 nm, the injection volume was 10 μl, the injection temperature was 15°C, and the spiked test solution was injected for testing.

[0071] Gradient elution program:

[0072] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 25 82 18 45 40 60 46 100 0 65 100 0

[0073] Detection spectrum see Figure 4 As shown, the retention time of methionylglycine in the spiked test solution is 9.779 min, and the resolution is greater than 1.40, which meets the requirements; the retention time of phenylethylamine is 21.413 min, and the resolution is greater than 1.40, which meets the requirements; the retention time of benzoic acid is 35.763 min, and the resolution is greater than 1.40, which meets the requirements; the retention time of phenylpropionic acid is 39.504 min.

[0074] Example 5

[0075] The difference between this embodiment and embodiment 4 is that the flow rate is 0.9 ml / min.

[0076] Detection spectrum see Figure 5 As shown, the retention time of methionylglycine in the spiked test solution is 10.760min, and the resolution is greater than 1.40, which meets the requirements; the retention time of phenylethylamine is 22.563min, and the resolution is greater than 1.40, which meets the requirements; the retention time of benzoic acid is 36.829min, and the resolution is greater than 1.40, which meets the requirements; the retention time of phenylpropionic acid is 40.509min.

[0077] Example 6

[0078] The difference between this embodiment and embodiment 4 is that the mobile phase A is a phosphoric acid aqueous solution with a concentration of 22.5 g / L.

[0079] Detection spectrum see Figure 6 As shown, the retention time of methionylglycine in the spiked test solution is 9.507 min, and the resolution is greater than 1.40, which meets the requirements; the retention time of phenylethylamine is 21.112 min, and the resolution is greater than 1.40, which meets the requirements; the retention time of benzoic acid is 35.616 min, and the resolution is greater than 1.40, which meets the requirements; the retention time of phenylpropionic acid is 39.387 min.

[0080] Example 7

[0081] The difference between this embodiment and embodiment 4 is that the mobile phase A is a phosphoric acid aqueous solution with a concentration of 23.5 g / L.

[0082] Detection spectrum see Figure 7As shown, the retention time of methionylglycine in the spiked test solution is 9.523min, and the resolution is greater than 1.40, which meets the requirements; the retention time of phenylethylamine is 21.147min, and the resolution is greater than 1.40, which meets the requirements; the retention time of benzoic acid is 35.629min, and the resolution is greater than 1.40, which meets the requirements; the retention time of phenylpropionic acid is 39.397min.

[0083] Test Example 1

[0084] System suitability experiment:

[0085] Methionylglycine positioning solution: Accurately measure 1 ml of methionylglycine reference stock solution, place it in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0086] Phenylethylamine positioning solution: Accurately measure 1 ml of phenethylamine reference stock solution, place it in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0087] Benzoic acid positioning solution: Accurately measure 1 ml of benzoic acid reference stock solution, place it in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0088] Phenylpropionic acid positioning solution: Accurately measure 1 ml of phenylpropionic acid reference stock solution, place it in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0089] Sensitivity solution: Accurately measure 2 ml of reference solution into a 10 ml volumetric flask, dilute to the mark with water, and shake well.

[0090] The methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid positioning solutions, reference solution, sensitivity solution and spiked test solution were respectively subjected to HPLC detection under the same detection conditions as in Example 4. The chromatograms are shown in FIG. Figure 8 As shown, the peak times of methionylglycine, phenethylamine, benzoic acid and phenylpropionic acid, as well as the minimum separation between methionylglycine, phenethylamine, benzoic acid and phenylpropionic acid and the adjacent peaks were obtained. The test results are as follows:

[0091]

[0092] As can be seen from the figure, in the blank solution and the negative blank solution, there are no interfering peaks at the peak positions of methionylglycine, phenethylamine, benzoic acid and phenylpropionic acid; the peaks of other components in the test solution have no interference at the peak positions of methionylglycine, phenethylamine, benzoic acid and phenylpropionic acid. As can be seen from the table, the minimum separation between methionylglycine, phenethylamine, benzoic acid and phenylpropionic acid and the adjacent component peaks in the spiked test solution is 1.41, which meets the requirement that the separation between the impurity peak and the adjacent component peak is greater than 1.2. The signal-to-noise ratio S / N of methionylglycine in the sensitivity solution is 13.1, which meets the requirement that the signal-to-noise ratio S / N is not less than 10.

[0093] In summary, the system applicability of this detection method is good.

[0094] Test Example 2

[0095] Specificity experiment:

[0096] The test sample is a destruction solution: accurately measure 2 ml of this product (i.e., compound amino acid injection (18AA-VII), the same below) and place it in a 10 ml volumetric bottle, dilute it to the scale with water, shake well, and obtain.

[0097] Acid degradation solution: Accurately measure 10 ml of this product and place it in a 50 ml volumetric flask, add 0.5 ml of 1 mol / L HCl solution, let it stand for 2 hours, then add the same volume of 1 mol / L NaOH solution to neutralize it, and dilute it to the scale with water, shake well, and you have it.

[0098] Acid degradation negative blank solution: Accurately measure 10 ml of negative blank solution, and the rest of the operation is the same as the acid degradation solution.

[0099] Alkali degradation solution: Accurately measure 10 ml of this product, add 0.5 ml of 1 mol / L NaOH solution, let it stand for 2 hours, then add the same volume of 1 mol / L HCl solution to neutralize it, dilute it to the scale with water, shake well, and you have it.

[0100] Alkaline degradation negative blank solution: Accurately measure 10 ml of negative blank solution, and the rest of the operation is the same as the alkaline degradation test solution.

[0101] Oxidative degradation solution: Accurately measure 10 ml of this product, place it in a 50 ml volumetric flask, introduce an appropriate amount of oxygen (about 5 minutes), leave it for 5 hours, and dilute it to the scale with water, shake well, and you have it.

[0102] Oxidative degradation negative blank solution: Accurately measure 10 ml of negative blank solution, and the rest of the operation is the same as the oxidative degradation test solution.

[0103] High temperature degradation solution: Accurately measure 10 ml of this product, place it in a 50 ml volumetric flask, place it in an 80°C water bath for 3 hours, cool it, dilute it to the scale with water, shake it well, and you have it.

[0104] High temperature degradation negative blank solution: Take 10ml of negative blank solution, and the rest of the operation is the same as that of high temperature degradation test solution.

[0105] Photodegradation solution: Take this product and place it under a light intensity of 5000Lx±500Lx for 10 days. Accurately measure 2ml and place it in a 10ml volumetric bottle. Dilute it to the scale with water and shake well.

[0106] Photodegradation negative blank solution: Take the negative blank, place it under a light intensity of 5000Lx±500Lx for 10 days, accurately measure 2ml and place it in a 10ml volumetric bottle, dilute it to the scale with water, shake well, and you have it.

[0107] The above 11 solutions were subjected to HPLC detection under the same detection conditions as in Example 4. The chromatograms are shown in FIG. Fig. 9 As shown in the figure, it can be seen that the blank solution, negative blank solution, and each forced degradation blank solution have no interference at the peak positions of methionylglycine, phenethylamine, benzoic acid, and phenylpropionic acid; in the chromatograms of each forced degradation test solution, the impurity peaks degraded do not interfere with the detection of methionylglycine, phenethylamine, benzoic acid, and phenylpropionic acid.

[0108] In summary, the detection method has good specificity, and the test solution is relatively stable under conditions of short-term illumination, strong acid, strong alkali, oxidation, and high temperature.

[0109] Test Example 3

[0110] Limit of quantitation and limit of detection experiments:

[0111] Quantitation limit solution: Take appropriate amount of methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid reference substance stock solutions respectively, and continue to dilute until the S / N (signal-to-noise ratio) is between 10 and 30.

[0112] Detection limit solution: Take appropriate amount of methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid reference substance stock solutions respectively, and continuously dilute until the S / N (signal-to-noise ratio) is between 3 and 10.

[0113] 1. Detect the concentrations of the quantitative limit solution and the detection limit solution, and calculate the percentage of the quantitative limit concentration to the limit concentration (2.1 μg / ml). The data are as follows:

[0114]

[0115] The quantitative limit concentration / limit concentration of each impurity is in the range of 7.0% to 20.6%, all meeting the requirement of less than 30%.

[0116] 2. Take 6 portions of the quantitative limit solution, and test the 6 portions of the quantitative limit solution and 1 portion of the detection limit solution respectively. The detection method is the same as that in Example 4. Calculate the relative standard deviation RSD of the peak area and retention time of the 6 portions of the quantitative limit solution after detection. The peak area statistics of the quantitative limit solution are as follows:

[0117] name Peak area-1 Peak area-2 Peak area-3 Peak area-4 Peak area-5 Peak area-6 RSD% Methionylglycine 0.0766 0.0796 0.0708 0.0779 0.0832 0.0794 5.3 Phenylethylamine 0.0969 0.0996 0.1062 0.1120 0.0920 0.1046 7.0 benzoic acid 0.0683 0.0722 0.0649 0.0702 0.0650 0.0664 4.4 Phenylpropionic acid 0.0755 0.0804 0.0857 0.0779 0.0848 0.0850 5.2

[0118] The peak area RSD of the quantitative limit solution for six consecutive injections was in the range of 4.4% to 7.0%, all meeting the requirement of less than 10.0%.

[0119] 3. Detect 6 quantitative limit solutions and 1 detection limit solution to obtain the signal-to-noise ratio S / N. The results of the signal-to-noise ratio S / N are as follows:

[0120]

[0121] The S / N of the detection limit of each impurity is 3.6 to 5.5, all within the required range of 3 to 10. The S / N of the quantitative limit solution of each impurity is between 10.3 and 28.1, all within the required range of 10 to 30.

[0122] In summary, the detection method has good sensitivity.

[0123] Test Example 4

[0124] Linearity experiment:

[0125] Quantitation limit solution: same as in Test Example 3.

[0126] 50% linear solution (1 μg / ml): Accurately measure 1 ml of the mixed impurity stock solution, place it in a 20 ml volumetric flask, and dilute to the scale with water.

[0127] 80% linear solution (1.6 μg / ml): Accurately measure 2 ml of the mixed impurity stock solution, place it in a 25 ml volumetric flask, and dilute to the scale with water.

[0128] 100% linear solution (2 μg / ml): Accurately measure 2 ml of the mixed impurity stock solution, place it in a 20 ml volumetric flask, and dilute to the mark with water.

[0129] 150% linear solution (3 μg / ml): Accurately measure 3 ml of the mixed impurity stock solution, place it in a 20 ml volumetric flask, and dilute to the scale with water.

[0130] 200% linear solution (4 μg / ml): Accurately measure 2 ml of the mixed impurity stock solution, place it in a 10 ml volumetric flask, and dilute to the mark with water.

[0131] Wherein, the preparation method of the mixed impurity stock solution is shown in Example 1.

[0132] The above solutions were subjected to liquid chromatography analysis and detection, and the concentrations and peak areas of methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid therein were detected and calculated, and the linear equations and correlation coefficients were calculated respectively. The data are as follows:

[0133]

[0134] Linear relationship diagram Fig.10 As shown, R 2 R in the above table is r. It can be seen from the above table that each known impurity shows good linearity in the range of quantitative limit to 200% linear solution, and the correlation coefficient r is in the range of 0.9997 to 0.9999, which meets the requirement of correlation coefficient r ≥ 0.999; and the Y-axis intercept / 100% limit concentration response value is in the range of -1.8% to 0.6%, which does not exceed the requirement of ±10%; at the same time, the linear relationship between the concentration of each known impurity and the peak area in this detection method is good.

[0135] Test Example 5

[0136] Accuracy experiment:

[0137] 50% recovery solution: Accurately measure 2 ml of the test solution and 0.5 ml of the mixed impurity stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with water, and shake well. Prepare 3 copies in parallel.

[0138] 100% recovery solution: Accurately measure 2 ml of the test solution and 1 ml of the mixed impurity stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with water, and shake well. Prepare 3 copies in parallel.

[0139] 150% recovery solution: Accurately measure 2 ml of the test solution and 1.5 ml of the mixed impurity stock solution, place them in the same 10 ml volumetric flask, dilute to the mark with water, and shake well. Prepare 3 copies in parallel.

[0140] The above 9 solutions were tested by high performance liquid chromatography, and the recovery rate of each impurity in the 9 solutions was calculated by external standard method, and the average value and RSD (relative standard deviation) were calculated. The test results are as follows:

[0141]

[0142]

[0143] As can be seen from the table above, the recovery rates of each impurity at the three concentration levels are in the range of 95.84% to 106.60%, which are all within the recovery rate requirement of 90.0% to 110.0%; the recovery rate of each concentration level and the recovery rate RSD of all concentration levels are in the range of 0.2% to 4.7%, which are all less than the recovery rate RSD requirement of 10.0%. In summary, the accuracy of this detection method is good.

[0144] Test Example 6

[0145] Stability test:

[0146] 1. Place the reference solution at 15°C, perform liquid chromatography detection and separation on the reference solution at 0h, 3h, 10h, 16.5h, 26.5h and 41.5h, and perform statistics on the peak areas in the chromatogram. The peak area statistics are as follows:

[0147]

[0148] It can be seen from the table that within 41.5 hours, the peak area RSD of each impurity was between 0.7% and 3.5%, all less than 5.0%, that is, the test results of the reference solution placed at 15°C for one day were stable.

[0149] 2. Place the spiked test solution at 15°C, perform liquid chromatography detection and separation on the spiked test solution at 0h, 3h, 10h, 16.5h, 26.5h, 37h and 67h, and perform statistics on the contents of methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid. The statistical results are as follows:

[0150]

[0151] It can be seen from the table that within 67 hours, the RSDs of the methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid detected in each time period were between 0.8% and 3.8%, all less than 10.0%, that is, the test results of the spiked test solution placed at 15°C for 2 days were stable.

[0152] Test Example 7

[0153] Durability test:

[0154] Sensitivity solution: Accurately measure 2 ml of reference solution and place it in a 10 ml volumetric flask, dilute to the mark with water, and shake well.

[0155] 1. When the flow rate was reduced by 0.1 ml / min and the concentration of phosphoric acid solution in mobile phase A was 22.5-23.5 g / L, different instruments, different chromatographic columns and injectors were replaced without temperature control, and the recovery rate of blank solution and spiked test solution was tested. The test results are as follows:

[0156]

[0157] Blank solution: Under other chromatographic conditions, there is no interference at the peak positions of methionylglycine, phenylethylamine, benzoic acid and phenylpropionic acid in the chromatograms of the blank solution and the negative sample solution.

[0158] Spiked test solution: Calculated by external standard method, the spiked recoveries of methionylglycine, phenylethylamine and benzoic acid were 96.61% to 104.03%, which were within the standard recoveries of 90.0% to 110.0%. The maximum RSD was 3.4%, which was within the standard RSD ≤ 10.0%, where na means not obtained.

[0159] 2. When the flow rate is reduced by 0.1 ml / min and the concentration of phosphoric acid solution in mobile phase A is 22.5-23.5 g / L, different instruments, different chromatographic columns and injectors are replaced without temperature control, and methionylglycine in the sensitivity solution is detected and the signal-to-noise ratio S / N is calculated. The calculation results are as follows:

[0160]

[0161] It can be seen from the table that the minimum signal-to-noise ratio of the methionylglycine peak is 12.6, which meets the standard that the signal-to-noise ratio should be no less than 10.

[0162] In summary, the method has good durability when the flow rate is reduced by 0.1 ml / min, the mobile phase A is ± 0.5 g / L, different instruments and chromatographic columns are replaced, and the injector is not temperature controlled.

[0163] In summary, the detection method provided by the present invention has a high degree of separation for impurities introduced or generated by glycine and phenylalanine, can effectively control the content of methionylglycine and / or phenylethylamine and / or benzoic acid and / or phenylpropionic acid in the injection, and improves the safety of the injection; at the same time, conventional reagents are used in the detection process, which is environmentally friendly, the detection method is simple and low-cost, suitable for practical application and promotion, and has broad application prospects.

[0164] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for detecting impurities in a compound amino acid injection, characterized in that: The detection method adopts high performance liquid chromatography, with octadecylsilane bonded silica gel as the chromatographic column, phosphoric acid solution with a concentration of 20.0-25.0 g / L as mobile phase A, methanol solution as mobile phase B, and the gradient elution procedure is as follows: The impurities include methionylglycine and / or phenethylamine and / or benzoic acid and / or phenylpropionic acid.

2. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: The gradient elution procedure is: 。 3. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: The concentration of the phosphoric acid solution is 22.5-23.5 g / L.

4. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: During the elution process, the flow rate of the mobile phase is 0.5-1.5 ml / min.

5. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: The injection volume is 3-20 μl.

6. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: The detection wavelength is 200~220nm.

7. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: The chromatographic column is Welch Ultimate LPAQ C18, 4.6 mm×250 mm, and the particle size of the silica gel particles is 5 μm.

8. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: The compound amino acid injection comprises glycine and / or phenylalanine.

9. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: The model of the compound amino acid injection is 18AA-VII.

10. The method for detecting impurities in the compound amino acid injection according to claim 1, characterized in that: The detection method is as follows: the chromatographic column model specification is Ultimate LP-AQ 4.6mm×250mm, 5μm, the column temperature is 30°C; the mobile phase A is a phosphoric acid solution with a concentration of 23.0g / L, the mobile phase B is methanol, the injection volume is 10μl, the flow rate is 1.0ml / min, and the detection wavelength is 205nm; the gradient elution program is as follows: 。

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