A detection method and separation method of vasopressin missing peptide

Abstract

The present invention relates to a detection method and a separation method of a vasopressin-deficient peptide, and the separation method of the vasopressin-deficient peptide comprises the following steps: subjecting a test liquid to chromatographic separation; the test liquid comprises vasopressin and vasopressin-deficient peptide; the chromatographic separation adopts a chromatographic column based on T3 bonding technology, the mobile phase of the chromatographic separation comprises phase A and phase B, and the elution condition satisfies: at 0-4 min, the volume ratio of phase A to phase B is 100:0, at 10-15 min, the volume ratio of phase A to phase B is 70:30-90:10, and at 15.1-20 min, the volume ratio of phase A to phase B is 100:0. The analysis time of the present invention is short, and the retention time of the vasopressin-deficient peptide can be long and the separation degree is high.

Description

Technical Field

[0001] The invention relates to a detection method and a separation method of vasopressin missing peptide. Background Art

[0002] Vasopressin is a nonapeptide drug composed of 9 amino acids with an amino acid structure of C—Y—F—Q—N—C—P—R—G—NH2 (SEQ ID NO: 3). The first amino acid forms a disulfide bond with the sixth cysteine, and the carboxyl group on the ninth glycine forms an amide. This drug is used to treat central diabetes insipidus, the initial stage of polyuria after brain surgery or head trauma, and abdominal muscle relaxation that is ineffective with other drugs. During the synthesis process of vasopressin, it is difficult to ensure that the raw materials are completely reacted, which will lead to the production of vasopressin deletion peptides. For example, the vasopressin deletion peptide after the tyrosine (abbreviated as Y) Tyr in the structure of vasopressin is deleted has an amino acid structure of C—F—Q—N—C—P—R—G—NH2 (SEQ ID NO: 1), the first position forms a disulfide bond with the fifth cysteine, and the carboxyl group on the eighth glycine forms an amide, or the vasopressin deletion peptide after the phenylalanine (abbreviated as F) Phe in the structure of vasopressin is deleted has an amino acid structure of C—Y—Q—N—C—P—R—G—NH2 (SEQ ID NO: 2), the first position forms a disulfide bond with the fifth cysteine, and the carboxyl group on the eighth glycine forms an amide. Both deletion peptides are difficult to retain and cannot be detected on reversed-phase liquid chromatography.

[0003] Drugs with the same structure are not included in the 2020 edition of the Chinese Pharmacopoeia. The United States Pharmacopoeia (USP-NF2024: Vasopressin) includes the vasopressin content detection and analysis method, and the Japanese Pharmacopoeia (JP18, Supplement Ⅰ) includes the vasopressin injection related substance analysis method. However, both methods cannot effectively separate all missing peptides of vasopressin. In the vasopressin detection methods of the United States Pharmacopoeia (USP-NF2024: Vasopressin) and the Japanese Pharmacopoeia (JP18, Supplement Ⅰ), the missing peptides overlap with the solvent peak and cannot be detected, and the analysis time is relatively long. Summary of the invention

[0004] The present invention solves the problem that vasopressin deletion peptides cannot be effectively separated in the prior art, and provides a detection method and a separation method for vasopressin deletion peptides. The method has a short analysis time, can make the retention time of vasopressin deletion peptides long, and have a high degree of separation.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for separating vasopressin-deficient peptides, which comprises the following steps: subjecting a test solution to chromatographic separation; the test solution comprises vasopressin and vasopressin-deficient peptides; the vasopressin-deficient peptides comprise vasopressin-deficient peptides in which a tyrosine fragment in the structure of vasopressin is deleted or vasopressin-deficient peptides in which a phenylalanine fragment in the structure of vasopressin is deleted;

[0007] The chromatographic separation uses a chromatographic column based on T3 bonding technology. The mobile phase of the chromatographic separation includes phase A and phase B. The elution conditions meet the following requirements:

[0008] At 0-4 min, the volume ratio of phase A to phase B was 100:0.

[0009] At 10-15 min, the volume ratio of phase A to phase B is always between 70:30 and 90:10.

[0010] Wherein, the A phase is a buffered saline solution and a first polar aprotic solvent in a volume ratio of 87: 13 to 95: 5; the concentration of the buffered saline solution is 10-100 mM, and the pH is 2.5-5.0; and the B phase is a second polar aprotic solvent.

[0011] In the present invention, the vasopressin deletion peptide is a vasopressin deletion peptide Vas[-Tyr 2 ] and the vasopressin peptide Vas[-Phe 3 ] In addition to the above, it may also include vasopressin-deficient peptides in which other amino acid fragments in the structure of vasopressin are deleted.

[0012] In the present invention, the test solution may further include a diluent. The diluent is conventionally selected in the art, and may be an acetic acid aqueous solution, for example, an acetic acid aqueous solution with a concentration of 0.25% (V / V).

[0013] In the present invention, in the test solution, the ratio of the mass of the vasopressin to the mass of the vasopressin deletion peptide can be 100: (1-2).

[0014] In the present invention, the concentration of vasopressin in the test solution may be 20-40 μg / mL.

[0015] In the present invention, the concentration of the vasopressin deletion peptide in the test solution may be 0.4-0.8 μg / mL.

[0016] In a specific embodiment of the present invention, the test solution includes: 40 μg / mL vasopressin, 0.4 μg / mL Vas[-Tyr 2 ], Vas[-Phe3 ], the diluent is an aqueous solution of acetic acid with a concentration of 0.25% (V / V).

[0017] In the present invention, the length of the chromatographic column may be 100-250 mm, preferably 150-250 mm.

[0018] In the present invention, the separation mode of the chromatographic column may be reverse phase.

[0019] In a specific embodiment of the present invention, the chromatographic column is an octadecyl-modified silica gel chromatographic column bonded based on T3 technology.

[0020] In the present invention, in the elution conditions, at 10-15 min, the volume ratio of phase A to phase B is preferably constant at a value between 80:20 and 90:10, for example, constant at 70:30, 80:20, 85:15, 88:12 or 90:10.

[0021] In the present invention, in the elution conditions, at 15.1-20 min, the volume ratio of phase A to phase B is 100:0.

[0022] In the present invention, the buffered salt solution is a conventional choice in the art, such as a diammonium hydrogen phosphate aqueous solution.

[0023] In the present invention, the concentration of the buffered saline solution is conventionally selected in the art, preferably 20-100 mM, such as 50 mM.

[0024] In the present invention, the pH of the buffered saline solution is conventionally selected in the art, preferably 2.5-4.0, such as 2.5, 3.0, 4.0 or 5.0.

[0025] In the present invention, the first polar aprotic solvent is selected conventionally in the art, such as acetonitrile.

[0026] In the present invention, the second polar aprotic solvent is conventionally selected in the art, such as acetonitrile.

[0027] In the present invention, during the chromatographic separation, the column temperature is conventionally selected in the art, preferably 30-45°C, more preferably 30-40°C, such as 30°C, 35°C, 40°C or 45°C.

[0028] In the present invention, during the chromatographic separation, the flow rate of the mobile phase is conventionally selected in the art, preferably 0.8-1.5 ml / min, more preferably 0.8-1.2 ml / min, such as 0.8 ml / min, 1.0 ml / min, 1.2 ml / min or 1.5 ml / min.

[0029] In the present invention, during the chromatographic separation, the injection volume is conventionally selected in the art, preferably 5-100 μl, more preferably 10-100 μl, such as 50 μl.

[0030] In the present invention, during the chromatographic separation, the detection wavelength is conventionally selected in the art, preferably 210-230 nm, more preferably 210-220 nm, such as 220 nm.

[0031] In the present invention, the chromatographic separation conditions may satisfy any one of the following conditions 1 to 14:

[0032] Condition 1: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 90:10 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0033] Condition 2: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0034] Condition three: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 85:15 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0035] Condition 4: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 80:20 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0036] Condition 5: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 70:30 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0037] Condition six: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 30°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0038] Condition seven: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 35°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0039] Condition eight: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 45°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0040] Condition nine: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 0.8 ml / min, and the injection volume is 50 μL.

[0041] Condition 10: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.2 ml / min, and the injection volume is 50 μL.

[0042] Condition 11: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.5 ml / min, and the injection volume is 50 μL.

[0043] Condition 12: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=2.5, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0044] Condition 13: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile in a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=4.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0045] Condition 14: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile in a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=5.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

[0046] In a second aspect, the present invention provides a method for detecting vasopressin deletion peptide, comprising the following steps: subjecting a second test liquid to chromatographic separation; the second test liquid comprises vasopressin;

[0047] The chromatographic separation uses a chromatographic column based on T3 bonding technology. The mobile phase of the chromatographic separation includes phase A and phase B. The elution conditions meet the following requirements:

[0048] At 0-4 min, the volume ratio of phase A to phase B was 100:0.

[0049] At 10-15 min, the volume ratio of phase A to phase B is always between 70:30 and 90:10.

[0050] Wherein, the A phase is a buffered saline solution and a first polar aprotic solvent in a volume ratio of 87: 13 to 95: 5; the concentration of the buffered saline solution is 10-100 mM, and the pH is 2.5-5.0; and the B phase is a second polar aprotic solvent.

[0051] In the present invention, the second test liquid may further include a diluent, which is preferably an acetic acid aqueous solution, for example, an acetic acid aqueous solution with a volume concentration of 0.25%.

[0052] In the present invention, the concentration of vasopressin in the second test solution may be 20-40 μg / mL.

[0053] In the present invention, the conditions for the chromatographic separation may be as described above.

[0054] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0055] The reagents and raw materials used in the present invention are commercially available.

[0056] The positive and progressive effects of the present invention are:

[0057] (1) In the present invention, the chromatographic column based on T3 bonding technology suitable for the retention of polar substances is selected to prolong the retention time of the missing peptides, especially the two weakly retained missing peptides Vas[-Tyr 2 ] and Vas[-Phe 3 ];

[0058] (2) In the present invention, by changing the mobile phase elution conditions, the peak position of the vasopressin deletion peptide can be optimized, and the deletion peptides (especially the two weakly retained deletion peptides Vas[-Tyr 2 ] and Vas[-Phe 3 ]) and vasopressin, and the analysis time can be shortened to about 20 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a comparison chart of HPLC chromatograms of vasopressin and two deletion peptides in Example 1.

[0060] Figure 2 This is a comparison chart of HPLC chromatograms of vasopressin and two missing peptides under different ratios of mobile phase in Example 2.

[0061] Figure 3 This is the ultraviolet full-wavelength scanning diagram of vasopressin VAS in Example 3.

[0062] Figure 4 This is a comparison of HPLC chromatograms of vasopressin and two missing peptides at different chromatographic column temperatures in Example 4.

[0063] Figure 5 This is a comparison chart of HPLC chromatograms of vasopressin and two deletion peptides under different flow rate conditions in Example 5.

[0064] Figure 6 This is a comparison chart of HPLC chromatograms of vasopressin and two deletion peptides under different mobile phase pH conditions in Example 6.

[0065] Figure 7 Vas[-Phe 3 ]Specificity positioning HPLC chromatogram of reference sample.

[0066] Figure 8 For Vas[-Tyr 2 ]Specificity positioning HPLC chromatogram of reference sample.

[0067] Fig. 9 This is a comparison chart of HPLC chromatograms of vasopressin and two missing peptides in Comparative Example 1.

[0068] Fig.10 This is a comparison chart of HPLC chromatograms of vasopressin and two missing peptides in Comparative Example 2. DETAILED DESCRIPTION

[0069] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0070] In the following examples, the raw materials and basic experimental conditions used are as follows:

[0071] Vas[-Tyr 2 ] Reference substance, batch number: 44A200401, was obtained from Shanghai First Biochemical Pharmaceutical Co., Ltd.

[0072] Vas[-Phe 3 ] Reference substance, batch number: 44A200817, from Shanghai First Biochemical Pharmaceutical Co., Ltd.

[0073] Vas reference substance was obtained from Shanghai First Pharmaceuticals Co., Ltd. In other embodiments, the following can also be purchased: USP United States Pharmacopoeia vasopressin standard substance, item number: 1711100, batch number: R150S0.

[0074] Diammonium hydrogen phosphate, from Sinopharm Chemical Reagent Co., Ltd.

[0075] Acetonitrile, from Fisher.

[0076] Phosphoric acid, from TEDIA.

[0077] Acetic acid, from TEDIA.

[0078] Liquid chromatograph, model: Waters Arc HPLC.

[0079] Example 1

[0080] 1. Solution preparation:

[0081] a. Dilution: 0.25% (V / V) acetic acid aqueous solution;

[0082] b. Mixed solution stock solution: weigh Vas[-Tyr 2 ] and Vas[-Phe 3] 4 mg of each reference substance was placed in the same 100 ml volumetric flask, dissolved in diluent and diluted to the scale to prepare a solution containing 0.04 mg of each known vasopressin deletion peptide per 1 ml.

[0083] c. Mixed solution of vasopressin and vasopressin deletion peptide: Weigh 4 mg of vasopressin working reference substance and place it in a 100 ml volumetric flask. Measure 1 ml of stock solution A and place it in the same 100 ml volumetric flask. Add diluent to dissolve and dilute to the scale to prepare a solution containing 40 μg of vasopressin and 0.4 μg of each vasopressin deletion peptide per 1 ml.

[0084] 2. Chromatographic separation:

[0085] The mixed solution of vasopressin and vasopressin-deficient peptide was subjected to chromatographic separation. The chromatographic conditions were as shown in Tables 1-1 to 1-2:

[0086] Table 1-1 Basic information

[0087]

[0088] Table 1-2 Elution gradient

[0089]

[0090] 3. Chromatogram Figure 1 As shown, according to the software Empower 3 Software Pro, Vas[-Phe 3 ] and Vas[-Tyr 2 ] is 28.8, Vas[-Tyr 2 ] and vasopressin was 24.8.

[0091] Example 2: Different ratios of aqueous phase and organic phase

[0092] The second stage of gradient elution is a strong elution stage, in which the aqueous phase and the organic phase are important conditions specially selected in this embodiment.

[0093] Experiment 2-1: Compared with Example 1, the only difference is that in the strong elution stage of 10 min to 15 min, the volume ratio of phase A to phase B is 100:0.

[0094] Experiment 2-2: Compared with Example 1, the only difference is that in the strong elution stage of 10 min to 15 min, the volume ratio of phase A to phase B is 90:10.

[0095] Experiment 2-3: Compared with Example 1, the only difference is that in the strong elution stage of 10 min to 15 min, the volume ratio of phase A to phase B is 85:15.

[0096] Experiment 2-4: Compared with Example 1, the only difference is that in the strong elution stage of 10 min to 15 min, the volume ratio of phase A to phase B is 80:20.

[0097] Experiment 2-5: Compared with Example 1, the only difference is that in the strong elution stage of 10 min to 15 min, the volume ratio of phase A to phase B is 70:30.

[0098] Table 2-0 shows the separation of vasopressin and two missing peptides at different ratios of aqueous phase to organic phase in this example. Figure 2 In the strong elution phase, the ratio of phase A to phase B was in the range of 70:30 to 90:10, and the separation of each vasopressin deletion peptide was good.

[0099] Table 2-0 Separation of vasopressin missing peptides at different mobile phase ratios

[0100]

[0101] Example 3: Detection of wavelength changes

[0102] Compared with Example 1, the only difference is that the detection wavelength is between 210 nm and 240 nm.

[0103] like Figure 3 When the detection wavelength increases from 210 nm to 240 nm, the ultraviolet absorption of each component decreases rapidly. Taking into account the absorption interference of organic solvents and buffer salts in the mobile phase at low ultraviolet wavelengths, the appropriate detection wavelength range is 210-230 nm.

[0104] Example 4: Different column temperatures

[0105] Experiment 4-1: Compared with Example 1, the only difference is that the temperature of the chromatographic column is 30°C.

[0106] Experiment 4-2: Compared with Example 1, the only difference is that the temperature of the chromatographic column is 35°C.

[0107] Experiment 4-3: The conditions are the same as those in Example 1.

[0108] Experiment 4-4: Compared with Example 1, the only difference is that the chromatographic column temperature is 45°C.

[0109] Table 4-1 and Table 4-2 show the separation of vasopressin and two missing peptides at different column temperatures in this example. The chromatograms are shown in Figure 4 When the column temperature was between 30 and 45 °C, the separation of the vasopressin deletion peptides was good.

[0110] Table 4-1 Theoretical plate numbers of vasopressin-deficient peptides at different column temperatures

[0111]

[0112] Table 4-2 Separation of vasopressin deletion peptides at different column temperatures

[0113]

[0114] Example 5: Different mobile phase flow rates

[0115] Experiment 5-1: Compared with Example 1, the only difference is that the flow rate of the mobile phase is 0.8 ml / min.

[0116] Experiment 5-2: Same conditions as in Example 1.

[0117] Experiment 5-3: Compared with Example 1, the only difference is that the flow rate of the mobile phase is 1.2 ml / min.

[0118] Experiment 5-4: Compared with Example 1, the only difference is that the flow rate of the mobile phase is 1.5 ml / min.

[0119] Table 5-1 and Table 5-2 show the separation of vasopressin and two missing peptides at different chromatographic column temperatures in this example. The chromatograms are shown in Figure 5 , the flow rate of the mobile phase was in the commonly used range of 0.8-1.5 ml / min, and the separation of each vasopressin deletion peptide was good.

[0120] Table 5-1 Theoretical plate number of vasopressin deletion peptide under different flow rate conditions

[0121]

[0122] Table 5-2 Separation of vasopressin missing peptides at different flow rates

[0123]

[0124] Example 6: Different pH values ​​of mobile phase buffer solutions

[0125] Experiment 6-1: Compared with Example 1, the only difference is that the pH of the diammonium hydrogen phosphate aqueous solution is 2.5.

[0126] Experiment 6-2: Same conditions as Example 1.

[0127] Experiment 6-3: Compared with Example 1, the only difference is that the pH of the diammonium hydrogen phosphate aqueous solution is 4.0.

[0128] Experiment 6-4: Compared with Example 1, the only difference is that the pH of the diammonium hydrogen phosphate aqueous solution is 5.0.

[0129] Table 6-0 shows the separation of vasopressin and two missing peptides at different column temperatures in this example. The chromatograms are shown in Figure 6 , the pH of the mobile phase buffer salt solution was in the range of 2.5 to 5.0, and the vasopressin deletion peptides were well separated.

[0130] Table 6-0 Separation of vasopressin deletion peptides at different mobile phase buffer solution pH

[0131]

[0132] Comparative Example 1: Vas[-Phe 3 ]Specificity positioning of reference substances

[0133] 1. Sample solution: Each 1 ml contains Vas[-Phe 3 ] 0.04 mg of solution.

[0134] 2. Chromatographic conditions are the same as in Example 1

[0135] 3. Chromatogram Figure 7 shown.

[0136] Comparative Example 2: Vas[-Tyr 2 ]Specificity positioning of reference substances

[0137] 1. Sample solution: Each 1 ml contains Vas[-Tyr 2 ] 0.04 mg of solution.

[0138] 2. Chromatographic conditions are the same as in Example 1

[0139] 3. Chromatogram Figure 8 shown.

[0140] Comparative Example 1: Detection method of vasopressin content in the United States Pharmacopoeia (USP-NF2024: Vasopressin)

[0141] 1. Sample injection: same as in Example 1.

[0142] 2. Chromatographic conditions: octadecylsilane bonded silica gel as filler (4.6×250 mm); 0.66wt% diammonium hydrogen phosphate aqueous solution (pH 3.0)-acetonitrile (87: 13, V / V) as mobile phase, isocratic elution for about 60 min; flow rate is 1.0 ml / min; column temperature is 40℃; detection wavelength is 220 nm; injection volume is 20 μl.

[0143] 3. Chromatogram Fig. 9 shown.

[0144] Comparative Example 2: Analysis of Related Substances in Japanese Pharmacopoeia (JP18, Supplement Ⅰ) Vasopressin Injection

[0145] 1. Sample injection: same as in Example 1.

[0146] 2. Chromatographic conditions: octadecylsilane bonded silica gel as filler (particle size 3 μm, 4.6×150 mm); 0.66wt% diammonium phosphate aqueous solution (pH 3.0)-acetonitrile (95: 5, V / V) as mobile phase A; 0.66wt% diammonium phosphate aqueous solution (pH 3.0)-acetonitrile (45: 55, V / V) as mobile phase B; flow rate 0.6 ml / min; column temperature 40℃; detection wavelength 220 nm; injection volume 20 μl. Gradient elution, 0~45 min, mobile phase A: B (90: 10, V / V), 90~100min, mobile phase A: B (30: 70, V / V).

[0147] 3. Chromatogram Fig.10 shown.

[0148] Table 7 shows the effect comparison between Examples 1-6 and Comparative Examples 1-2, as follows:

[0149] Table 7

[0150]

[0151] Within the appropriate chromatographic conditions of each embodiment of the present invention, Vas[-Phe 3 ] and Vas[-Tyr 2 ] The separation degree is greater than 19, Vas[-Tyr 2 ] and vasopressin have a separation degree greater than 13. 3 ] retention time range 4.0-7.2 min, Vas[-Tyr 2 The retention time range of 5-6-nitropropene was 9.7-13.8 min, the retention time range of vasopressin was 13.7-18.0 min, there was no interference from the solvent peak, and the method had good specificity.

[0152] In summary, the present invention compares the retention behaviors of two highly polar missing peptides, investigates the suitable range of conditions such as chromatographic columns, mobile phase elution conditions, flow rate, temperature, pH, injection volume, etc., so that the main component and the missing peptide, the missing peptide and the solvent peak are both separated with good separation. The establishment of the HPLC analysis method of vasopressin missing peptide improves the purity of the drug process intermediates and the accuracy of the test results of related substances in the finished product, greatly shortens the test time, and can be used for product quality control and finished product testing in the drug production process.

Claims

1. A method for isolating vasopressin-deficient peptides, characterized in that: The method comprises the following steps: subjecting a test solution to chromatographic separation; the test solution comprises vasopressin and vasopressin-deficient peptide; the vasopressin-deficient peptide comprises a vasopressin-deficient peptide in which a tyrosine fragment in the structure of vasopressin is deleted or a vasopressin-deficient peptide in which a phenylalanine fragment in the structure of vasopressin is deleted; The chromatographic separation uses a chromatographic column based on T3 bonding technology. The mobile phase of the chromatographic separation includes phase A and phase B. The elution conditions meet the following requirements: At 0-4 min, the volume ratio of phase A to phase B was 100:

0. At 10-15 min, the volume ratio of phase A to phase B is always a value between 70:30 and 90:

10. Wherein, the A phase is a buffered saline solution and a first polar aprotic solvent in a volume ratio of 87: 13 to 95: 5; the concentration of the buffered saline solution is 10-100 mM, and the pH is 2.5-5.0; and the B phase is a second polar aprotic solvent.

2. The method for isolating vasopressin-deficient peptide according to claim 1, characterized in that: It meets at least one of the following conditions: (1) The test solution also includes a vasopressin-deficient peptide in which other amino acid fragments in the structure of vasopressin are deleted; (2) The test solution further includes a diluent; the diluent is preferably an acetic acid aqueous solution, for example, an acetic acid aqueous solution with a volume concentration of 0.25%; (3) In the test solution, the concentration of vasopressin is 20-40 μg / mL; (4) In the test solution, the concentration of the vasopressin deletion peptide is 0.4-0.8 μg / mL.

3. The method for isolating vasopressin-deficient peptide according to claim 2, characterized in that: The test solution includes: vasopressin at a concentration of 40 μg / mL, vasopressin deletion peptide with a concentration of 0.4 μg / mL after the tyrosine fragment in the structure of vasopressin is deleted, and vasopressin deletion peptide with a concentration of 0.4 μg / mL after the phenylalanine fragment in the structure of vasopressin is deleted. The diluent is an acetic acid aqueous solution with a volume concentration of 0.25%.

4. The method for isolating vasopressin-deficient peptide according to claim 1, characterized in that: The chromatographic separation satisfies at least one of the following conditions: (1) The length of the chromatographic column is 100-250 mm, preferably 150-250 mm, for example 250 mm; (2) The separation mode of the chromatographic column is reverse; (3) The chromatographic column is an octadecyl-modified silica gel chromatographic column bonded based on T3 technology; (4) During the chromatographic separation, the column temperature is 30-45°C, preferably 30-40°C, such as 30°C, 35°C, 40°C or 45°C; (5) During the chromatographic separation, the flow rate of the mobile phase is 0.8-1.5 ml / min, preferably 0.8-1.2 ml / min, for example 0.8 ml / min, 1.0 ml / min, 1.2 ml / min or 1.5 ml / min; (6) During the chromatographic separation, the injection volume is 5-100 μl, preferably 10-100 μl, for example 50 μl; (7) During the chromatographic separation, the detection wavelength is 210-230 nm, preferably 210-220 nm, for example 220 nm.

5. The method for isolating vasopressin-deficient peptide according to claim 1, characterized in that: The chromatographic separation satisfies at least one of the following conditions: (1) The buffered saline solution is an aqueous solution of diammonium hydrogen phosphate; (2) The concentration of the buffered saline solution is 20-100 mM, for example, 50 mM; (3) The pH of the buffered saline solution is 2.5-4.0, for example, 2.5, 3.0, 4.0 or 5.0; (4) In the elution conditions, at 10-15 min, the volume ratio of phase A to phase B is always a value between 80:20 and 90:10, for example, always 70:30, 80:20, 85:15, 88:12 or 90:10; (5) In the elution conditions, at 15.1-20 min, the volume ratio of phase A to phase B is 100:0; (6) The first polar aprotic solvent is acetonitrile; (7) The second polar aprotic solvent is acetonitrile.

6. The method for isolating vasopressin-deficient peptide according to claim 1, characterized in that: The chromatographic separation satisfies any one of the following conditions 1 to 14: Condition 1: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 90:10, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 2: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88:12, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is a 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 3: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 85:15, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is a 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 4: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 80: 20, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 5: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 70: 30, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 6: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88: 12, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 30°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 7: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88: 12, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 35°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 8: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88: 12, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 45°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition nine: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88: 12, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 0.8 ml / min, and the injection volume is 50 μL; Condition 10: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88: 12, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.2 ml / min, and the injection volume is 50 μL; Condition 11: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88: 12, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=3.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.5 ml / min, and the injection volume is 50 μL; Condition 12: The chromatographic column is an octadecyl modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88: 12, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=2.5, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 13: The chromatographic column is an octadecyl modified silica gel chromatographic column bonded based on T3 technology, the length of the chromatographic column is 250 mm, at 10.00-15.00 min, the volume ratio of phase A to phase B is 88: 12, phase A is a buffer solution and acetonitrile with a volume ratio of 95: 5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=4.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL; Condition 14: The chromatographic column is an octadecyl-modified silica gel column bonded based on T3 technology, the length of the chromatographic column is 250 mm, the volume ratio of phase A to phase B is 88:12 at 10.00-15.00 min, phase A is a buffer solution and acetonitrile with a volume ratio of 95:5, the buffer solution is 50 mM diammonium hydrogen phosphate aqueous solution, pH=5.0, phase B is acetonitrile, the column temperature is 40°C, the detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 ml / min, and the injection volume is 50 μL.

7. A method for detecting vasopressin-deficient peptide, characterized in that: The method comprises the following steps: subjecting a second test liquid to chromatographic separation; the second test liquid comprises vasopressin; The chromatographic separation uses a chromatographic column based on T3 bonding technology. The mobile phase of the chromatographic separation includes phase A and phase B. The elution conditions meet the following requirements: At 0-4 min, the volume ratio of phase A to phase B was 100:

0. At 10-15 min, the volume ratio of phase A to phase B is always a value between 70:30 and 90:

10. Wherein, the A phase is a buffered saline solution and a first polar aprotic solvent in a volume ratio of 87: 13 to 95: 5; the concentration of the buffered saline solution is 10-100 mM, and the pH is 2.5-5.0; and the B phase is a second polar aprotic solvent.

8. The method for detecting vasopressin deficiency peptide according to claim 7, characterized in that: The second test liquid satisfies at least one of the following conditions: (1) The second test liquid further includes a diluent; the diluent is preferably an acetic acid aqueous solution, for example, an acetic acid aqueous solution with a volume concentration of 0.25%; (2) In the second test solution, the concentration of vasopressin is 20-40 μg / mL.

9. The method for detecting vasopressin deficiency peptide according to claim 7, characterized in that: The conditions for the chromatographic separation are as defined in claim 5 or 6.

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