A high performance liquid chromatography analysis method for O-sulfo-L-tyrosine sodium salt and its related substances

Abstract

The present invention provides a high performance liquid chromatography analysis method for O-sulfo-L-tyrosine sodium salt and related substances. Specifically, a high performance liquid chromatography method for analyzing O-sulfo-L-tyrosine sodium salt and related substances is provided. The method of the present invention has a stable chromatogram baseline, a reasonable peak time, good peak symmetry and good reproducibility, and is very suitable for the qualitative and quantitative detection of O-sulfo-L-tyrosine sodium salt.

Description

Technical Field

[0001] The invention relates to a high performance liquid chromatography analysis method for O-sulfo-L-tyrosine sodium salt and related substances. Background Art

[0002] O-Sulfo-L-tyrosine sodium salt, CAS No.: 98930-06-4, molecular formula C 9 H 10 NO 6 S·Na has a molecular weight of 283.23 and is an off-white or yellow powder with the structural formula:

[0003]

[0004] O-sulfo-L-tyrosine sodium salt is an amino acid compound used in proteomics research. The sulfonation of tyrosine in organisms is a common phenomenon. Therefore, tyrosine-O-sulfonic acid has a wide range of applications in peptide and protein synthesis. The introduction of sulfonic acid with a strong acid group greatly improves the hydrophilicity of tyrosine. The introduction of tyrosine-O-sulfonic acid in peptide synthesis can improve the water solubility of peptides to a certain extent, which is beneficial to the subsequent peptide synthesis and purification.

[0005] The synthesis process of O-sulfo-L-tyrosine sodium salt has been gradually improved under continuous improvement, but there are no relevant reports or relevant literature on the detection method. In the synthesis process of chemically synthesized O-sulfo-L-tyrosine sodium salt, it is inevitable to introduce a series of impurities, and their presence will seriously affect the product quality of O-sulfo-L-tyrosine sodium salt. Introduce unnecessary impurities for subsequent polypeptide synthesis. Therefore, in order to ensure the product quality of O-sulfo-L-tyrosine sodium salt and improve safety, it is of great significance to strictly control the quality of its related substances. Summary of the invention

[0006] The purpose of the present invention is to provide a high performance liquid chromatography analysis method for efficiently and quantitatively detecting O-sulfo-L-tyrosine sodium salt and its related substances.

[0007] Specifically, a high performance liquid chromatography analysis method for O-sulfo-L-tyrosine sodium salt and related substances is provided, comprising the steps of:

[0008] (i) providing a sample;

[0009] (ii) loading the sample onto a high performance liquid chromatograph for analysis to obtain chromatographic analysis data, wherein the chromatographic detection conditions include:

[0010] Chromatographic column: C8 column, 150mm×4.6mm, 3.5μm;

[0011] The mobile phase consists of mobile phase A and mobile phase B;

[0012] Mobile phase A: 0.01% to 0.1% v / v trifluoroacetic acid aqueous solution;

[0013] Mobile phase B: volume ratio of acetonitrile: water: trifluoroacetic acid = (450-600): (350-650): (0-1);

[0014] Elution mode: gradient elution; and

[0015] The detection wavelength is 200-230nm.

[0016] In another preferred embodiment, the mobile phase A is 0.04%-0.1% v / v trifluoroacetic acid aqueous solution, more preferably 0.05-0.08% v / v trifluoroacetic acid aqueous solution.

[0017] In another preferred embodiment, the volume ratio of water, acetonitrile and trifluoroacetic acid in mobile phase B is (480-520):(480-520):(0.4-0.6), and more preferably the volume ratio of water, acetonitrile and trifluoroacetic acid is 500:500:0.5.

[0018] In another preferred embodiment, the mobile phase A is a 0.05% v / v trifluoroacetic acid aqueous solution; the volume ratio of water, acetonitrile and trifluoroacetic acid in the mobile phase B is 500:500:0.5.

[0019] In another preferred embodiment, the dilution solvent of the sample is water.

[0020] In another preferred embodiment, the method includes one or more of the following features:

[0021] The column temperature is 25-50°C, preferably 35-45°C, more preferably 40°C.

[0022] The flow rate is 0.5-1.2 mL / min, preferably 0.8-1.2 mL / min, more preferably 1.0 mL / min;

[0023] The injection volume is 5-20 μL, preferably 10-15 μL; and / or

[0024] The UV detection wavelength is 210-220 nm, preferably 214 nm.

[0025] In another preferred embodiment, the stationary phase of the chromatographic column is octylsilane bonded silica gel (LP-C8), and more preferably the chromatographic column is Ultimate LP-C8, 150 mm×4.6 mm, 3.5 μm.

[0026] In another preferred embodiment, the chromatograph is E2695 (Waters).

[0027] In another preferred embodiment, the elution gradient is:

[0028]

[0029]

[0030] In another preferred embodiment, the elution gradient is:

[0031]

[0032] In another preferred embodiment, the method includes: chromatograph: E2695 (Waters) chromatographic column: Ultimate LP-C8 150 mm×4.6 mm, 3.5 μm

[0033] Mobile phase A: 0.5% or 0.1% v / v trifluoroacetic acid

[0034] Mobile phase B: water, acetonitrile, trifluoroacetic acid volume ratio of 500:500:0.5; flow rate: 1.0ml / min

[0035] Column temperature: 40°C

[0036] Injection volume: 10ul

[0037] Detection wavelength: 214nm

[0038] Elution gradient:

[0039]

[0040]

[0041] In another preferred embodiment, the related impurities include L-tyrosine. Using the method of the present invention, L-tyrosine peaks within 0.5-1 min after the main peak, and its peak shape and separation degree meet the determination requirements.

[0042] In another preferred embodiment, the method further comprises the step of: using a standard sample to perform qualitative analysis on the sample by retention time.

[0043] In another preferred embodiment, the method further comprises the step of: using a standard sample to establish an external standard curve by the method, so as to quantitatively analyze the concentration of the analyte in the sample to be tested.

[0044] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is the spectrum of the gradient program containing an organic phase in the initial ratio in Example 2;

[0046] Figure 2 is the spectrum of the gradient program without organic phase in the initial ratio in Example 2;

[0047] Figure 3 is the spectrum of Example 3 with 50% acetonitrile as mobile phase B;

[0048] Figure 4 is the spectrum of 50% acetonitrile containing 0.05% TFA as mobile phase B in Example 3;

[0049] Figure 5 This is a spectrum of Example 4. DETAILED DESCRIPTION

[0050] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, have provided a high performance liquid chromatography method for analyzing O-sulfo-L-tyrosine sodium salt and its related substances. The method of the invention has a stable chromatogram baseline, a reasonable peak time, good peak symmetry and good reproducibility, and is very suitable for qualitative and quantitative detection of O-sulfo-L-tyrosine sodium salt. The present invention was completed on this basis.

[0051] the term

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0053] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0054] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0055] HPLC analysis method

[0056] A high performance liquid chromatography analysis method for O-sulfo-L-tyrosine sodium salt and related substances, characterized in that it comprises the steps of:

[0057] (i) providing a sample;

[0058] (ii) loading the sample onto a high performance liquid chromatograph for analysis to obtain chromatographic analysis data, wherein the chromatographic detection conditions include:

[0059] Chromatographic column: C8 column, 150mm×4.6mm, 3.5μm;

[0060] The mobile phase consists of mobile phase A and mobile phase B;

[0061] Mobile phase A: 0.01% to 0.1% v / v trifluoroacetic acid aqueous solution;

[0062] Mobile phase B: volume ratio of acetonitrile: water: trifluoroacetic acid = (450-600): (350-650): (0-1);

[0063] Elution mode: gradient elution; and

[0064] The detection wavelength is 200-230nm.

[0065] Preferably, the elution gradient is:

[0066]

[0067] Preferably, the chromatographic conditions include:

[0068] Instrument: E2695 (Waters)

[0069] Column: UltimateLP-C8 150mm×4.6mm, 3.5μm

[0070] Mobile phase A: 0.01% to 0.1% trifluoroacetic acid aqueous solution

[0071] Mobile phase B: water: acetonitrile: trifluoroacetic acid = (350-650): (450-600): (0-1) (volume ratio)

[0072] Flow rate: 0.5~1.2ml / min

[0073] Column temperature: 25~50℃

[0074] Injection volume: 10ul

[0075] Detection wavelength: 214nm

[0076] Elution gradient:

[0077]

[0078] Usually, the related impurities include L-tyrosine. Using the method of the present invention, L-tyrosine peaks within 0.5-1 min after the main peak, and its peak shape and separation degree meet the determination requirements.

[0079] Furthermore, the method further comprises the step of: using a standard sample to perform a qualitative analysis on the sample by retention time.

[0080] Furthermore, the method further comprises the step of: using a standard sample to establish an external standard curve through the method, thereby quantitatively analyzing the concentration of the analyte in the sample to be tested.

[0081] The main advantages of the present invention include:

[0082] The invention provides a high performance liquid chromatography method for analyzing O-sulfo-L-tyrosine sodium salt and related substances thereof. The method of the invention has a stable chromatogram baseline, a reasonable peak time, good peak symmetry and good reproducibility, and is very suitable for qualitative and quantitative detection of O-sulfo-L-tyrosine sodium salt.

[0083] The present invention will be further described below in conjunction with specific implementation. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0084] Example 1

[0085] Test solution: Take about 10 mg of O-sulfo-L-tyrosine sodium salt sample, dissolve it in pure water as solvent and make up to volume as the test solution.

[0086] Instrument and chromatographic conditions:

[0087] Instrument: E2695 (Waters)

[0088] Column: UltimateLP-C8 150mm×4.6mm, 3.5μm

[0089] Mobile phase A: Condition changes

[0090] Mobile phase B: water: acetonitrile: trifluoroacetic acid = 500: 500: 0 (volume ratio)

[0091] Flow rate: 1.0ml / min

[0092] Column temperature: 40°C

[0093] Injection volume: 10ul

[0094] Detection wavelength: 214nm

[0095] Elution gradient:

[0096]

[0097] Using 0.05% and 0.1% trifluoroacetic acid aqueous solution as mobile phase A respectively, other conditions remained unchanged, and the chromatograms were recorded.

[0098] Experimental conclusion:

[0099] When the above two concentrations of trifluoroacetic acid are used as the mobile phase, the peak shape symmetry and other aspects meet the measurement requirements, but the pH of 0.1% trifluoroacetic acid is about 1.5-2.5, while the pH of 0.05% trifluoroacetic acid is about 2.0-3.5. In order to avoid the loss of filler in the chromatographic column in an overly acidic environment, 0.05% trifluoroacetic acid is used as mobile phase A.

[0100] Example 2

[0101] Instrument and chromatographic conditions:

[0102] Instrument: E2695 (Waters)

[0103] Column: UltimateLP-C8 150mm×4.6mm, 3.5μm

[0104] Mobile phase A: 0.1% trifluoroacetic acid

[0105] Mobile phase B: water: acetonitrile: trifluoroacetic acid = 500: 500: 0 (volume ratio)

[0106] Flow rate: 1.0ml / min

[0107] Column temperature: 40°C

[0108] Injection volume: 10ul

[0109] Detection wavelength: 214nm

[0110] Elution gradient: Conditional variable phase

[0111] The following two gradients were used for detection respectively, and the chromatograms were recorded while other conditions remained unchanged:

[0112] Gradient 1:

[0113]

[0114] Gradient 2:

[0115]

[0116] Experimental conclusion:

[0117] When the initial ratio of gradient 1 contains mobile phase B, the mobile phase contains about 5% acetonitrile. Due to the strong elution ability of acetonitrile, the main peak appears too early (e.g. Figure 1 ), and the solvent peak is concentrated in the front, which makes it impossible to perform quantitative detection. Therefore, gradient 2 is selected as the experimental gradient (e.g. Figure 2 ).

[0118] Example 3

[0119] Instrument and chromatographic conditions:

[0120] Instrument: E2695 (Waters)

[0121] Column: UltimateLP-C8 150mm×4.6mm, 3.5μm

[0122] Mobile phase A: 0.1% trifluoroacetic acid

[0123] Mobile phase B: Condition changes

[0124] Flow rate: 1.0ml / min

[0125] Column temperature: 40°C

[0126] Injection volume: 10ul

[0127] Detection wavelength: 214nm

[0128] Elution gradient:

[0129]

[0130] Using 50% acetonitrile solution and 50% acetonitrile solution containing 0.05% trifluoroacetic acid as mobile phase B respectively, and recording the chromatograms under the condition that other conditions remain unchanged.

[0131] Experimental conclusion:

[0132] When mobile phase B does not contain 0.05% trifluoroacetic acid, the baseline fluctuates greatly during the gradient change (e.g. Figure 3 ), which brings great interference to quantitative detection. After adding 0.05% trifluoroacetic acid, the baseline is relatively stable, which is conducive to quantitative detection. Therefore, 50% acetonitrile containing 0.05% trifluoroacetic acid was selected as mobile phase B (such as Figure 4 ).

[0133] The present invention studies the influence of mobile phase A, mobile phase B and elution gradient on the result.

[0134] The experiment of Example 1 shows that when mobile phase A is 0.05% TFA, the mobile phase pH range is better, the damage to the machine and the chromatographic column is small, and the peak shape symmetry is not much different from that of 0.1% TFA. Therefore, 0.05% TFA is selected as mobile phase A.

[0135] Example 4

[0136] Instrument and chromatographic conditions:

[0137] Instrument: E2695 (Waters)

[0138] Column: UltimateLP-C8 150mm×4.6mm, 3.5μm

[0139] Mobile phase A: 0.05% trifluoroacetic acid

[0140] Mobile phase B: 50% acetonitrile solution containing 0.05% trifluoroacetic acid

[0141] Flow rate: 1.0ml / min

[0142] Column temperature: 40°C

[0143] Injection volume: 10ul

[0144] Detection wavelength: 214nm

[0145] Elution gradient:

[0146]

[0147] from Figure 5 It can be seen that with 0.05% trifluoroacetic acid as mobile phase A and 50% acetonitrile solution containing 0.05% trifluoroacetic acid as mobile phase B, the chromatogram has a stable baseline, a reasonable peak time, good peak symmetry and good reproducibility, which is very suitable for the qualitative and quantitative detection of O-sulfo-L-tyrosine sodium salt. And the separation between the main peak and the impurities before and after the detection method (such as L-tyrosine peaks about 0.5min after the main peak, its separation from the main peak, peak shape, etc. meet the determination requirements), theoretical plate number and tailing factor can meet the method requirements.

[0148] The experiment in Example 2 shows that the initial gradient must not contain an organic phase, otherwise the peak elution time is too early, resulting in the inability to completely separate the main peak from the solvent peak, affecting the detection results. Therefore, the C8 column Yuexu Ultimate LP-C8, which is resistant to low pH and pure water phase washing, is selected as the chromatographic column of this method. Therefore, the gradient program is adjusted to an initial ratio of pure water phase without an organic phase, and then the organic phase ratio is gradually increased after the main peak is washed out.

[0149] The experiment of Example 3 shows that when 50% acetonitrile solution is used as mobile phase B, the absorption values ​​vary greatly during the gradient process and the baseline is not stable. Therefore, 50% acetonitrile solution containing 0.05% trifluoroacetic acid is selected as mobile phase B of this method.

[0150] The experiment of Example 4 shows that under the experimental conditions of the present invention, the peak shape, baseline, reproducibility and separation degree from impurities of the chromatogram all have excellent effects, and the chromatographic conditions are mild and cause little damage to the chromatographic column.

[0151] The method of the present invention uses an octyl stationary phase filler chromatographic column, which has higher low pH tolerance and resistance to pure water phase washing, as well as lower carbon loading, and can more accurately and effectively determine O-sulfo-L-tyrosine sodium salt and related substances under specific chromatographic conditions. The separation degree between the main peak and the front and rear impurities, the theoretical plate number and the tailing factor of the detection method provided by the present invention can meet the method requirements.

[0152] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A high performance liquid chromatography analysis method for O-sulfo-L-tyrosine sodium salt and its related substances, characterized in that: Includes steps: (i) providing a sample; taking about 10 mg of O-sulfo-L-tyrosine sodium salt sample, dissolving it in pure water as a solvent and fixing the volume to obtain a test solution; (ii) loading the sample onto a high performance liquid chromatograph for analysis to obtain chromatographic analysis data, wherein the chromatographic detection conditions include: Chromatographic column: C8 column, 150mm×4.6mm, 3.5μm; The mobile phase consists of mobile phase A and mobile phase B; Mobile phase A: 0.01% to 0.1% v / v trifluoroacetic acid aqueous solution; Mobile phase B: the volume ratio of water, acetonitrile and trifluoroacetic acid is (480-520): (480-520): (0.4-0.6); Elution mode: gradient elution; and The detection wavelength is 200-230nm; The elution gradient is: Wherein, the related substance is L-tyrosine.

2. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The mobile phase A is 0.04%-0.1% v / v trifluoroacetic acid aqueous solution.

3. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The volume ratio of water, acetonitrile and trifluoroacetic acid in mobile phase B was 500:500:0.

5.

4. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The mobile phase A is a 0.05% v / v trifluoroacetic acid aqueous solution; the volume ratio of water, acetonitrile and trifluoroacetic acid in the mobile phase B is 500:500:0.

5.

5. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The method comprises one or more of the following features: The column temperature is 25-50°C; Flow rate: 0.5-1.2 mL / min; Injection volume is 5-20 μL; and / or The UV detection wavelength is 210-220nm.

6. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The chromatographic column is Ultimate LP-C8, 150 mm×4.6 mm, 3.5 μm.

7. The high performance liquid chromatography analysis method according to any one of claims 1 to 6, characterized in that: The method comprises one or more of the following features: The column temperature is 35-40°C; Flow rate: 0.8-1.0 mL / min; Injection volume is 10-15 μL; and / or The UV detection wavelength is 214nm.

8. The high performance liquid chromatography analysis method according to any one of claims 1 to 6, characterized in that: The elution gradient is:

9. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The method includes, chromatograph: E2695 Waters Column: UltimateLP-C8 150mm×4.6mm, 3.5μm Mobile phase A: 0.5% or 0.1% v / v trifluoroacetic acid Mobile phase B: water, acetonitrile, trifluoroacetic acid in a volume ratio of 500:500:0.5; Flow rate: 1.0ml / min Column temperature: 40°C Injection volume: 10ul Detection wavelength: 214nm Elution gradient:

10. The high performance liquid chromatography analysis method according to claim 1, characterized in that: The method further comprises the step of: using a standard sample to establish an external standard curve through the method, thereby quantitatively analyzing the concentration of the analyte in the sample to be tested.

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