Method for determining amino acid ratio of acetic acid diillegal forest
Through the treatment of short-term hydrolysis and pH adjustment, combined with pre-column derivatization and gradient elution technology, the problems of long hydrolysis time, excessive acidity and long elution time in the determination method for the ratio of amino acids of illicarone acetate in the prior art are solved. An efficient and accurate determination of amino acid ratios is achieved.
Patent Information
- Application Number
- CN202411919027.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-02-11
AI Technical Summary
In the prior art, the amino acid ratio measurement method for illefalin acetate in the amino acid ratio has the problems of long sample hydrolysis time, excessive acidity may damage the chromatographic column, and long elution time and low efficiency.
The sample was hydrolyzed for a short time and adjusted pH to neutral, combined with the pre-column derivatization treatment, and a octadecylsilane-bonded silica gel-filled chromatography column was set, and a gradient elution procedure was set to reduce the elution time.
The sample hydrolysis time is shortened, the damage to the chromatographic column is reduced, the separation between amino acids is improved, the sensitivity is high, the reproducibility is good, and the running time is short. It can accurately determine the ratio of amino acids in illicone acetate.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medical detection, and particularly relates to a method for determining the amino acid ratio of difacillin acetate. Background Art
[0002] Difaline acetate is a peripherally restricted and selective kappa opioid receptor agonist, mainly used to treat moderate to severe pruritus caused by chronic kidney disease (CKD) in adult patients undergoing hemodialysis. It effectively reduces the intensity of uremic pruritus by specifically activating kappa opioid receptors, thereby improving the quality of life of patients. As a raw material drug, the quality control of difaline acetate is crucial.
[0003] As an important indicator for the quality control of peptide drugs, the amino acid ratio can reflect the amino acid composition of the drug, thereby indirectly verifying the correctness of the amino acid sequence. Difacoumyl acetate is prepared by solid phase synthesis, which sequentially condenses amino acids according to the predetermined peptide sequence, followed by cleavage, purification, salt conversion and freeze-drying. In its production process, three amino acids, D-lysine, D-leucine and D-phenylalanine, are mainly used.
[0004] However, the quality standard of difacillin acetate is not recorded in the pharmacopoeias and related literatures of various countries. Therefore, developing an accurate and rapid method for determining the amino acid ratio is of great significance for ensuring the quality of difacillin acetate API.
[0005] In the Chinese Pharmacopoeia 2020, Part II, there are some problems with the determination method of the amino acid ratio of thymopentin. The method requires taking an appropriate amount of sample, adding hydrochloric acid solution (1→2), hydrolyzing at 110°C for 24 hours, and then using an appropriate amino acid analysis method for determination. However, the method does not clearly state the derivatization method and specific analysis method, and the sample is too acidic after hydrolysis with hydrochloric acid solution, which may cause serious damage to the chromatographic column, and the hydrolysis time is too long.
[0006] In addition, the amino acid determination method of the compound amino acid injection (18AA-III) uses a chromatographic column filled with octadecylsilane bonded silica gel. The injection sample does not need to be hydrolyzed and is directly derivatized and then detected under gradient elution conditions. However, the elution time is as long as 60 minutes, which is inefficient. Summary of the invention
[0007] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a method for determining the amino acid ratio of difacoum acetate. The hydrolysis time of the sample solution is short, and the pH is adjusted to neutral after hydrolysis, thereby reducing column damage; the separation between the amino acids is good, the sensitivity is high, the reproducibility is good, the running time is short, and the amino acid ratio in difacoum acetate can be accurately determined.
[0008] The method for determining the amino acid ratio of difacillin acetate of the present invention comprises the following steps: (1) Solution preparation a. Weigh an appropriate amount of acetic acid difaxian sample, add acid to hydrolyze it, adjust the pH to near neutral, add internal standard solution to prepare the test solution; b. Take D-lysine salt reference substance, D-leucine reference substance, and D-phenylalanine reference substance, add internal standard solution to prepare reference substance solution; (2) Determination Take the blank solution, test solution and reference solution respectively, perform the same derivatization treatment, inject the derivatized solutions into the liquid chromatograph respectively, record the chromatogram, and calculate the ratio of D-lysine, D-leucine and D-phenylalanine by the peak area according to the internal standard method; The specific steps of the derivatization treatment are: adding triethylamine acetonitrile solution to the blank solution, the test solution, and the reference solution, and then adding phenyl isothiocyanate acetonitrile solution, reacting in a water bath, and then cooling to room temperature, adding n-hexane, shaking, and leaving until clarified, and taking the clarified lower layer liquid as the derivatized solution.
[0009] The chromatographic conditions include: Detector: PDA or UV detector; Chromatographic column: Octadecylsilane bonded silica gel is used as filler, the chromatographic column is SHIMADZU ShimNex HEC18-AQ, the specification is 4.6mm×250mm, 5µm, or InerSustain AQ-C18, the specification is 4.6mm×250mm, 5µm; Detection wavelength: 245nm-254nm; Column temperature: 25-35°C; Mobile phase: acetate buffer solution-acetonitrile mixed solution is used as mobile phase A, and acetonitrile-water mixed solution is used as mobile phase B; the preparation method of the acetate buffer solution is to weigh 3.28-4.92g of sodium acetate, add water to dissolve and dilute, and adjust the pH value to 6.3-6.7 with acetic acid.
[0010] Elution method: gradient elution, as shown in Table 1, where % is volume percentage.
[0011] Table 1 Elution program
[0012] Flow rate: 0.9-1.1mL / min.
[0013] The acetate buffer solution is an acetic acid solution with a concentration of 0.04-0.06 mol / L and a pH of 6.3-6.7; the volume ratio of the acetate buffer solution to acetonitrile in the mobile phase A is 94:6-92:8, and the volume ratio of acetonitrile to water in the mobile phase B is 78:22-82:18.
[0014] The internal standard solution is prepared by weighing an appropriate amount of D-norleucine, adding water to dissolve and diluting to prepare an internal standard solution with a D-norleucine concentration of 40 μg / mL.
[0015] In the reference solution, the ratio of D-lysine to D-leucine is 0.8-1.2, and the ratio of D-phenylalanine is 1.6-2.4.
[0016] The reference solution is prepared by weighing appropriate amounts of a D-lysine salt reference substance, a D-leucine reference substance, and a D-phenylalanine reference substance, placing them in the same volumetric flask, adding an internal standard solution, dissolving them in water, and diluting them to prepare a reference solution. The concentrations of D-lysine, D-leucine, and D-norleucine in the reference solution are 40 μg / mL, and the concentration of D-phenylalanine is 96 μg / mL.
[0017] The calculation method of each amino acid is as follows: The D-lysine ratio=the mole number of D-lysine÷[(the mole number of D-lysine+the mole number of D-leucine+the mole number of D-phenylalanine)÷4]; the D-leucine ratio=the mole number of D-leucine÷[(the mole number of D-lysine+the mole number of D-leucine+the mole number of D-phenylalanine)÷4]; the D-phenylalanine ratio=the mole number of D-phenylalanine÷[(the mole number of D-lysine+the mole number of D-leucine+the mole number of D-phenylalanine)÷4].
[0018] The test solution is prepared by adding a difamoyl acetate sample to a hydrochloric acid solution and hydrolyzing it at 105-115° C. for 11-13 hours, cooling it to room temperature, adjusting the pH to 6.0-8.0, adding an internal standard solution, and diluting it with water to prepare a test solution with a difamoyl acetate concentration of 0.2 mg / mL.
[0019] In the derivatization treatment, the phenyl isothiocyanate acetonitrile solution is prepared by dissolving phenyl isothiocyanate in acetonitrile with a concentration of 0.1 mol / L-1 mol / L; the triethylamine acetonitrile solution is prepared by dissolving triethylamine in acetonitrile with a concentration of 0.1 mol / L-1 mol / L, the water bath reaction temperature is 40°C-50°C, and the water bath reaction time is 30min-35min.
[0020] The volume ratio of the blank solution, the test solution, and the reference solution to the triethylamine acetonitrile solution is 2:1; the volume ratio of the blank solution, the test solution, and the reference solution to the phenyl isothiocyanate acetonitrile solution is 2:1; the volume ratio of the blank solution, the test solution, and the reference solution to n-hexane is 2:1.
[0021] The blank solution is water.
[0022] In the step (2), 2 mL of blank solution water, test solution, and reference solution were taken respectively, placed in stoppered test tubes, 1 mL of 1 mol / L triethylamine acetonitrile solution and 1 mL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution were accurately added, shaken, reacted in a 45°C water bath for 35 min, taken out and cooled, and then 1 mL of n-hexane was accurately added respectively, shaken, and the solution was allowed to stand until it was clarified. The clarified lower layer liquid was taken and injected into a liquid chromatograph respectively, and the chromatogram was recorded.
[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) In the method for determining the amino acid ratio of difacoumyl acetate of the present invention, the sample hydrolysis time is short, and after the hydrolysis is completed, the pH of the solution is adjusted to neutral before sample injection and analysis, thereby reducing damage to the chromatographic column.
[0024] (2) The method for determining the amino acid ratio of difacoumyl acetate of the present invention uses a chromatographic column with octadecylsilane bonded silica gel as a filler and sets a gradient elution program. The elution time is short, the derivative blank does not interfere with the peaks of each amino acid, and the specificity is strong.
[0025] (3) The method for determining the amino acid ratio of difacoum acetate of the present invention has good separation between the amino acids, high sensitivity and good reproducibility, and can accurately determine the amino acid ratio in difacoum acetate. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is the HPLC chromatogram of the blank water after treatment in Example 1.
[0027] Figure 2 The HPLC chromatogram of the reference solution after treatment in Example 1.
[0028] Figure 3 The HPLC chromatogram of the test solution after treatment in Example 1.
[0029] Figure 4 The HPLC chromatogram of the test solution after treatment in Comparative Example 1.
[0030] Figure 5 It is the HPLC chromatogram of the test solution in Comparative Example 2.
[0031] Figure 6 It is the HPLC chromatogram of the blank water after treatment in Example 2.
[0032] Figure 7 The HPLC chromatogram of the reference solution after treatment in Example 2.
[0033] Figure 8 The HPLC chromatogram of the test solution after treatment in Example 2.
[0034] Fig. 9 It is the HPLC chromatogram of the blank water after treatment in Example 3.
[0035] Fig.10 The HPLC chromatogram of the reference substance solution after treatment in Example 3.
[0036] Fig.11 The HPLC chromatogram of the test solution after treatment in Example 3.
[0037] Fig.12 It is the HPLC chromatogram of the blank water after treatment in Example 4.
[0038] Fig.13 The HPLC chromatogram of the reference solution after treatment in Example 4.
[0039] Fig.14 The HPLC chromatogram of the test solution after treatment in Example 4.
[0040] Fig.15 It is the HPLC chromatogram of the blank water after treatment in Example 5.
[0041] Fig.16 It is the HPLC chromatogram of the reference substance solution after treatment in Example 5.
[0042] Fig.17 The HPLC chromatogram of the test solution after treatment in Example 5.
[0043] Fig.18 It is the HPLC chromatogram of the blank water after treatment in Example 6.
[0044] Fig.19 The HPLC chromatogram of the reference solution after treatment in Example 6.
[0045] Fig. 20 The HPLC chromatogram of the test solution after treatment in Example 6.
[0046] Fig.21 It is the HPLC chromatogram of the blank water after treatment in Example 7.
[0047] Fig. 22 The HPLC chromatogram of the reference solution after treatment in Example 7.
[0048] Fig.23 The HPLC chromatogram of the test solution after treatment in Example 7.
[0049] Fig.24 It is the HPLC chromatogram of the blank water after treatment in Example 8.
[0050] Fig.25 The HPLC chromatogram of the reference solution after treatment in Example 8.
[0051] Fig.26 The HPLC chromatogram of the test solution after treatment in Example 8.
[0052] Fig. 27 It is the HPLC chromatogram of the blank water after treatment in Example 9.
[0053] Fig.28 The HPLC chromatogram of the reference solution after treatment in Example 9.
[0054] Fig.29 The HPLC chromatogram of the test solution after treatment in Example 9.
[0055] Fig.30 It is the HPLC chromatogram of the blank water after treatment in Example 10.
[0056] Fig.31 The HPLC chromatogram of the reference solution after treatment in Example 10.
[0057] Fig.32 The HPLC chromatogram of the test solution after treatment in Example 10.
[0058] Fig.33 The HPLC chromatogram of the test solution after treatment in Example 11.
[0059] Fig.34 The HPLC chromatogram of the blank water after treatment in Example 12.
[0060] Fig.35 The HPLC chromatogram of the reference solution after treatment in Example 12.
[0061] Fig.36 The HPLC chromatogram of the test solution after treatment in Example 12.
[0062] Fig.37 The HPLC chromatogram of the blank water after treatment in Example 13.
[0063] Fig.38 The HPLC chromatogram of the reference solution after treatment in Example 13.
[0064] Fig.39 The HPLC chromatogram of the test solution after treatment in Example 13.
[0065] Fig.40 It is the HPLC chromatogram of the blank water after treatment in Example 14.
[0066] Fig.41 This is the HPLC chromatogram of the reference solution after treatment in Example 14.
[0067] Fig.42 This is the HPLC chromatogram of the test solution after treatment in Example 14.
[0068] Fig.43 It is the HPLC chromatogram of the blank water after treatment in Example 15.
[0069] Fig.44 This is the HPLC chromatogram of the reference solution after treatment in Example 15.
[0070] Fig.45 This is the HPLC chromatogram of the test solution after treatment in Example 15.
[0071] Fig.46 The HPLC chromatogram of the blank water after treatment in Example 16.
[0072] Fig.47 The HPLC chromatogram of the reference solution after treatment in Example 16.
[0073] Fig.48 This is the HPLC chromatogram of the test solution after treatment in Example 16. DETAILED DESCRIPTION
[0074] The present invention will be further described below in conjunction with specific embodiments.
[0075] The raw materials and reagents used in the following examples and comparative examples are all commercially available products. The hydrochloric acid used is commercially available concentrated hydrochloric acid with a concentration of 36%.
[0076] This product, Difalin Acetate: is produced by Shandong Qidu Pharmaceutical Co., Ltd.; D-norleucine: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; D-Lysine salt reference substance: purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd.; D-leucine reference substance: purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd.; D-phenylalanine reference substance: purchased from Shanghai Haohong Biotechnology Co., Ltd.
[0077] Example 1 The method for determining the amino acid ratio of difacoumyl acetate comprises the following steps: (1) Chromatographic conditions: Detector: PDA detector; Chromatographic column: SHIMADZU ShimNex HE C18-AQ, specification: 4.6mm×250mm, 5µm; Detection wavelength: 254nm; Column temperature: 30°C; Mobile phase: 0.05 mol / L acetate buffer salt and acetonitrile were mixed in a volume ratio of 93:7 as mobile phase A, and acetonitrile and water were mixed in a volume ratio of 80:20 as mobile phase B; the preparation method of acetate buffer salt was to weigh 4.1 g of sodium acetate, dissolve and dilute it with water, and adjust the pH value to 6.5 with dilute acetic acid.
[0078] The gradient program is shown in Table 1.
[0079] Flow rate: 1.0 mL / min; Injection volume: 10 μL; (2) Preparation of internal standard solution: Accurately weigh 20 mg of D-norleucine, place in a 10 mL volumetric flask, add water to dissolve and dilute to the scale, shake well.
[0080] (3) Preparation of reference solution: Accurately weigh 20 mg of D-lysine salt reference substance, 20 mg of D-leucine reference substance, and 48 mg of D-phenylalanine reference substance, place them in the same 10 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well; accurately measure 1 mL, place it in a 50 mL volumetric flask, accurately add 1 mL of internal standard solution, dilute to the scale with water, and shake well.
[0081] (4) Preparation of test solution: Accurately weigh 10 mg of difarline acetate, add 2 mL of hydrochloric acid solution (prepared with concentrated hydrochloric acid and water in a volume ratio of 1:1), hydrolyze at 110°C for 12 h, cool and adjust the pH to 7, transfer to a 50 mL volumetric flask, accurately add 1 mL of internal standard solution, dilute to the scale with water, and shake well.
[0082] (5) Determination method: Accurately measure 2 mL of water, test solution and reference solution, respectively, and place them in stoppered test tubes. Accurately add 1 mL of 1 mol / L triethylamine acetonitrile solution and 1 mL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution, shake well, and react in a water bath at 45°C for 35 minutes. Take out and cool, then accurately add 1 mL of n-hexane, shake well, and place the solution until it is clear. Take the clear lower layer liquid and inject it into the liquid chromatograph respectively, record the chromatogram, as shown in the figure below. Figure 1 , Figure 2 , Figure 3 As shown, the ratio of each amino acid was calculated using the peak area according to the internal standard method.
[0083] The 0.1 mol / L phenyl isothiocyanate acetonitrile solution is obtained by dissolving 0.1 mol of phenyl isothiocyanate in 1 L of acetonitrile; the 1 mol / L triethylamine acetonitrile solution is obtained by dissolving 1 mol of triethylamine in 1 L of acetonitrile.
[0084] Depend on Figure 1 , Figure 2 , Figure 3 It can be seen that the blank peak after derivatization does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution appear in sequence. The peak shape of each amino acid is good, the separation is good, and the ratio of each amino acid meets the requirements. The specific results are shown in Table 2.
[0085] The calculation formula of the amino acid ratio is: Correction factor ( f )= ; In the formula A S is the peak area of the internal standard substance; A R is the peak area of the amino acid in the reference substance; c S is the concentration of the internal standard substance (mg / mL); c R is the concentration of the corresponding amino acid in the reference substance (mg / mL).
[0086] content( c X )= f × ; In the formula A X is the peak area of the amino acid in the test sample; c x is the concentration of the corresponding amino acid in the test sample (mg / mL); A' S is the peak area of the internal standard substance; c' S is the concentration of the internal standard substance; f is the internal standard correction factor.
[0087] Amino Acid Ratio = ; In the formula c x is the concentration of amino acid in the test sample (mg / mL); M X is the molecular weight of the corresponding amino acid ( M D-赖氨酸 :146, M D-亮氨酸 :131, M D-苯丙氨酸 :165); is the total molar number of D-lysine, D-leucine, and D-phenylalanine in the test sample.
[0088] Table 2 Example 1 amino acid ratio results
[0089] Comparative Example 1 The chromatographic conditions, solution preparation and determination method of this comparative example are the same as those of Example 1, except that the elution gradient program is carried out according to the following Table 3.
[0090] Table 3 Comparative Example 1 Elution Procedure
[0091] The HPLC chromatogram of the test solution of this comparative example is as follows Figure 4 As shown in the figure, it can be seen that D-leucine, D-norleucine, D-phenylalanine and D-lysine all elute at 30 minutes and later, the retention time of D-phenylalanine and the adjacent impurity peaks are similar, and D-lysine and the adjacent impurity peaks are not baseline separated, indicating that this comparative example is not suitable for the determination of amino acid ratios in this product.
[0092] Comparative Example 2 The chromatographic conditions, other solution preparation and determination methods of this comparative example are the same as those of Example 1, except that the preparation of the test solution is replaced by the following steps: Accurately weigh 10 mg of difacillin acetate, place it in a 50 mL volumetric flask, accurately add 1 mL of internal standard solution, dilute to the scale with solvent, and shake well.
[0093] The HPLC chromatogram of the test solution of this comparative example is as follows Figure 5 As shown in the figure, it can be seen that there is only D-norleucine peak in the chromatogram of the test solution, while D-leucine, D-phenylalanine and D-lysine do not show peaks, indicating that these three amino acids are not free. This comparative example is not suitable for the determination of amino acid ratios in this product.
[0094] Example 2 This example is exactly the same as Example 1, except that the flow rate of 1.0 mL / min in the chromatographic conditions is replaced by 0.9 mL / min. After the measurement, the chromatogram is recorded.
[0095] The HPLC chromatograms of the blank aqueous solution, reference solution and test solution after treatment in this example are shown as follows: Figure 6-8 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 4.
[0096] Table 4 Example 2 amino acid ratio results
[0097] Example 3 This example is exactly the same as Example 2, except that the flow rate of 0.9 mL / min in the chromatographic conditions is replaced by 1.1 mL / min. After the measurement, the chromatogram is recorded.
[0098] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 9-11 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 5.
[0099] Table 5 Example 3 amino acid ratio results
[0100] Example 4 This embodiment is exactly the same as embodiment 1, except that the column temperature of 30° C. in the chromatographic conditions is replaced by 25° C. After the measurement, the chromatogram is recorded.
[0101] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 12-14As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 6.
[0102] Table 6 Example 4 amino acid ratio results
[0103] Example 5 This example is exactly the same as Example 4, except that the column temperature of 25° C. in the chromatographic conditions is replaced by 35° C. After the measurement, the chromatogram is recorded.
[0104] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 15-17 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 7.
[0105] Table 7 Example 5 amino acid ratio results
[0106] Example 6 This example is exactly the same as Example 1, except that the pH 6.5 of the acetate buffer in the chromatographic conditions is replaced with pH 6.3, and the chromatogram is recorded after the measurement.
[0107] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 18-20 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 8.
[0108] Table 8 Example 6 Amino Acid Ratio Results
[0109] Example 7 This example is exactly the same as Example 6, except that the pH 6.3 of the acetate buffer in the chromatographic conditions is replaced with pH 6.7, and the chromatogram is recorded after the measurement.
[0110] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 21-23 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 9.
[0111] Table 9 Example 7 Amino Acid Ratio Results
[0112] Example 8 This example is exactly the same as Example 1, except that the concentration of acetate buffer in the chromatographic conditions is replaced from 0.05 mol / L to 0.04 mol / L. After the measurement, the chromatogram is recorded.
[0113] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 24-26 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 10.
[0114] Table 10 Example 8 amino acid ratio results
[0115] Example 9 This example is exactly the same as Example 8, except that the concentration of acetate buffer in the chromatographic conditions is replaced from 0.04 mol / L to 0.06 mol / L. After the measurement, the chromatogram is recorded.
[0116] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 27-29 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 11.
[0117] Table 11 Example 9 amino acid ratio results
[0118] Example 10 This example is exactly the same as Example 1, except that the "SHIMADZUShimNex HE C18-AQ, specification 4.6mm×250mm, 5µm" in the chromatographic conditions is replaced by "InerSustain AQ-C18, specification 4.6mm×250mm, 5µm", the water bath temperature is replaced by "40°C" from "45°C", and the water bath time is replaced by "35min" from "30min". After the measurement, the chromatogram is recorded.
[0119] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 30-32 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 12.
[0120] Table 12 Example 10 Amino Acid Ratio Results
[0121] Embodiment 11 The method for determining the amino acid ratio of difacoumyl acetate comprises the following steps: (1) Chromatographic conditions: Detector: UV detector; Chromatographic column: SHIMADZU ShimNex HE C18-AQ, specification: 4.6mm×250mm, 5µm; Detection wavelength: 245nm; Column temperature: 30°C; Mobile phase: 0.05 mol / L acetate buffer and acetonitrile in a volume ratio of 93:7 as mobile phase A, acetonitrile and water in a volume ratio of 80:20 as mobile phase B; the preparation method of acetate buffer is to weigh 4.1 g of sodium acetate, dissolve and dilute it with water, and adjust the pH value to 6.5 with dilute acetic acid; The gradient program is shown in Table 1; Flow rate: 1.0 mL / min; Injection volume: 10 μL; (2) Preparation of test solution: Accurately weigh 10 mg of difacillin acetate, add 2 mL of hydrochloric acid solution (prepared with concentrated hydrochloric acid and water in a volume ratio of 1:1), hydrolyze at 105°C for 13 h, cool and adjust the pH to 8.0, transfer to a 50 mL volumetric flask, dilute to the mark with solvent, and shake well.
[0122] (3) Determination method: Accurately measure 2 mL of the test solution, place it in a stoppered test tube, accurately add 1 mL of 1 mol / L triethylamine acetonitrile solution, 1 mL of 0.1 mol / L phenyl isothiocyanate acetonitrile solution, shake well, and react in a water bath at 45°C for 35 minutes. Take it out and let it cool, then accurately add 1 mL of n-hexane, shake well, let the solution stand until it is clear, take the clear lower layer, inject it into the liquid chromatograph, and record the chromatogram.
[0123] The HPLC chromatogram of the test solution in this example is as follows Fig.33 As shown in the figure, it can be seen that D-leucine, D-phenylalanine and D-lysine peak in sequence, and the separation between the peaks is good. However, compared with the 254nm detection wavelength, the baseline fluctuation is larger at the 245nm detection wavelength.
[0124] Example 12 The chromatographic conditions and solution preparation of this embodiment are exactly the same as those of Example 1, with the difference being that the determination method of "precisely measuring water, 2 mL of the test solution and the reference solution, placing them in stoppered test tubes, accurately adding 1 mL of a 1 mol / L triethylamine acetonitrile solution and 1 mL of a 0.1 mol / L phenyl isothiocyanate acetonitrile solution, shaking well, and reacting in a water bath at 45°C for 35 min. Taking out and cooling, accurately adding 1 mL of n-hexane to each solution, shaking well, leaving the solution to clarify, and taking the clarified lower layer of liquid" is replaced by "precisely measuring water, 2 mL of the test solution and the reference solution, accurately placing them in stoppered test tubes, accurately adding 1 mL of a 0.1 mol / L triethylamine acetonitrile solution and 1 mL of a 1 mol / L phenyl isothiocyanate acetonitrile solution, shaking well, and reacting in a water bath at 50°C for 35 min. Taking out and cooling, accurately adding 1 mL of n-hexane to each solution, shaking well, leaving the solution to clarify, and taking the clarified lower layer of liquid". The 1 mol / L phenyl isothiocyanate acetonitrile solution is obtained by dissolving 1 mol of phenyl isothiocyanate in 1 L of acetonitrile; the 0.1 mol / L triethylamine acetonitrile solution is obtained by dissolving 0.1 mol of triethylamine in 1 L of acetonitrile.
[0125] In step (4) of the preparation of the test solution, "hydrolyze at 110°C for 12 h, cool and adjust the pH to 7.0" is replaced with "hydrolyze at 115°C for 13 h, cool and adjust the pH to 6.0".
[0126] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 34-36 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 13.
[0127] Table 13 Example 12 Amino Acid Ratio Results
[0128] Embodiment 13 This example is exactly the same as Example 1, except that the volume ratio of 0.05 mol / L acetate buffer and acetonitrile in mobile phase A in the chromatographic conditions is replaced from 93:7 to 92:8. After the measurement, the chromatogram is recorded. The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown below: Figure 37-39 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 14.
[0129] Table 14 Example 13 amino acid ratio results
[0130] Embodiment 14 This example is exactly the same as Example 13, except that the volume ratio of 0.05 mol / L acetate buffer and acetonitrile in mobile phase A in the chromatographic conditions is replaced from 92:8 to 94:6, and after measurement, the chromatogram is recorded.
[0131] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figure 40-42 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 15.
[0132] Table 15 Example 14 Amino Acid Ratio Results
[0133] Embodiment 15 This example is exactly the same as Example 1, except that the volume ratio of acetonitrile and water in mobile phase B in the chromatographic conditions is replaced from 80:20 to 78:22. After the measurement, the chromatogram is recorded. The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown below. Figure 43-45As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 16.
[0134] Table 16 Example 15 Amino Acid Ratio Results
[0135] Example 16 This example is exactly the same as Example 15, except that the volume ratio of acetonitrile and water in mobile phase B in the chromatographic conditions is replaced from 78:22 to 82:18, and after measurement, the chromatogram is recorded.
[0136] The HPLC chromatograms of the derived blank, reference solution and test solution in this example are shown in Figure 1. Figures 46-48 As shown in the figure, it can be seen that the derived blank peak does not interfere with the detection of the present invention. D-leucine, D-norleucine, D-phenylalanine and D-lysine in the reference solution and the test solution are sequentially peaked, and each amino acid has a good peak shape and good separation. The ratios of each amino acid meet the requirements, and the specific results are shown in Table 17.
[0137] Table 17 Example 16 Amino Acid Ratio Results
[0138] As can be seen from the above, the present invention adopts the method of first hydrolyzing the sample, adjusting the pH to neutral, and then pre-column derivatization, thereby reducing damage to the chromatographic column. Then, a chromatographic column with octadecylsilane bonded silica gel as a filler is used, and a reasonable and fast gradient elution program is set. The derivatization blank does not interfere with the peaks of each amino acid, has good specificity, and has good separation between each amino acid. The amino acid ratio in difacillin acetate can be accurately determined, thereby objectively, accurately and comprehensively evaluating the quality of difacillin acetate, which has important practical significance for product quality control.
Claims
1. A method for determining the amino acid ratio of difacoum acetate, characterized in that: The following steps are involved: (1) Solution preparation a. Weigh the acetic acid difaxian sample, add acid to hydrolyze it, adjust the pH to near neutral, add the internal standard solution to prepare the test solution; b. Take D-lysine salt reference substance, D-leucine reference substance, and D-phenylalanine reference substance, add internal standard solution to prepare reference substance solution; (2) Determination Take the blank solution, test solution and reference solution respectively, perform the same derivatization treatment, inject the derivatized solutions into the liquid chromatograph respectively, record the chromatogram, and calculate the ratio of D-lysine, D-leucine and D-phenylalanine by the peak area according to the internal standard method; The specific steps of the derivatization treatment are: adding triethylamine acetonitrile solution to the blank solution, the test solution, and the reference solution, and then adding phenyl isothiocyanate acetonitrile solution, reacting in a water bath, and then cooling to room temperature, adding n-hexane, shaking, and leaving until clarified, and taking the clarified lower layer liquid as the derivatized solution.
2. The method for determining the amino acid ratio of difacoum acetate according to claim 1, characterized in that: The chromatographic conditions include: Detector: PDA or UV detector; Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Detection wavelength: 245-254nm; Column temperature: 25-35°C; Mobile phase: Acetate buffer solution-acetonitrile mixed solution was used as mobile phase A, and acetonitrile-water mixed solution was used as mobile phase B; Elution mode: gradient elution; Flow rate: 0.9-1.1mL / min.
3. The method for determining the amino acid ratio of difacoum acetate according to claim 2, characterized in that: The gradient elution program is as follows: at 0 min, mobile phase A is 80% and mobile phase B is 20%; at 5 min, mobile phase A is 80% and mobile phase B is 20%; at 20 min, mobile phase A is 65% and mobile phase B is 35%; at 21 min, mobile phase A is 0% and mobile phase B is 100%; at 26 min, mobile phase A is 0% and mobile phase B is 100%; at 27 min, mobile phase A is 80% and mobile phase B is 20%; at 35 min, mobile phase A is 80% and mobile phase B is 20%.
4. The method for determining the amino acid ratio of difacoum acetate according to claim 2, characterized in that: The acetate buffer solution is a sodium acetate solution with a concentration of 0.04-0.06 mol / L and a pH of 6.3-6.7; The volume ratio of acetate buffer solution to acetonitrile in the mobile phase A is 94:6-92:8, and the volume ratio of acetonitrile to water in the mobile phase B is 78:22-82:
18.
5. The method for determining the amino acid ratio of difacoum acetate according to claim 1, characterized in that: The internal standard solution is a solution obtained by dissolving D-norleucine in water.
6. The method for determining the amino acid ratio of difacoum acetate according to claim 1, characterized in that: In the reference solution, the ratio of D-lysine to D-leucine is 0.8-1.2, and the ratio of D-phenylalanine is 1.6-2.
4.
7. The method for determining the amino acid ratio of difacillin acetate according to claim 1, characterized in that: The test solution is prepared by adding a difascic acid acetate sample to a hydrochloric acid solution and hydrolyzing it at 105-115° C. for 11-13 hours, cooling it to room temperature, adjusting the pH to 6.0-8.0, adding an internal standard solution, and diluting it with water to prepare the test solution.
8. The method for determining the amino acid ratio of difacoum acetate according to claim 1, characterized in that: In the derivatization treatment, the phenyl isothiocyanate acetonitrile solution is prepared by dissolving phenyl isothiocyanate in acetonitrile with a concentration of 0.1 mol / L-1 mol / L; the triethylamine acetonitrile solution is prepared by dissolving triethylamine in acetonitrile with a concentration of 0.1 mol / L-1 mol / L, the water bath reaction temperature is 40°C-50°C, and the water bath reaction time is 30min-35min.
9. The method for determining the amino acid ratio of difacoum acetate according to claim 8, characterized in that: The volume ratio of the blank solution, the test solution, and the reference solution to the triethylamine acetonitrile solution is 2:1; the volume ratio of the blank solution, the test solution, and the reference solution to the phenyl isothiocyanate acetonitrile solution is 2:1; the volume ratio of the blank solution, the test solution, and the reference solution to n-hexane is 2:
1.
10. The method for determining the amino acid ratio of difacoum acetate according to claim 1, characterized in that: The blank solution is water.