A liquid chromatography analysis method for bis(4-(octadecyloxy)phenyl)methanol

Abstract

The invention provides a kind of detection method of bis (4 ‑ (octadecyloxy) phenyl) methanol, uses liquid chromatography to detect the product containing bis (4 ‑ (octadecyloxy) phenyl) methanol, and its mobile phase consists of: tetrahydrofuran ‑ acetonitrile mixed solution and aqueous formic acid solution. By using tetrahydrofuran ‑ acetonitrile mixed solution as organic phase, the elution power of mobile phase relative to bis (4 ‑ (octadecyloxy) phenyl) methanol is increased, and detection result is accurate. In addition, the detection method of bis (4 ‑ (octadecyloxy) phenyl) methanol provided by the present invention is carried out methodological investigation, and experimental result shows that the detection method system of bis (4 ‑ (octadecyloxy) phenyl) methanol provided by the present invention has good applicability, strong specificity, good precision and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of chromatographic analysis, and particularly relates to a liquid chromatographic analysis method of bis(4-(octadecyloxy)phenyl)methanol. Background Art

[0002] Peptides are compounds formed by α-amino acids linked together by peptide bonds and are also intermediate products of protein hydrolysis. Many active substances in the human body exist in the form of peptides. Peptides are involved in the body's hormones, nerves, cell growth, and reproduction. Their importance lies in regulating the physiological functions of various systems and cells in the body, activating relevant enzymes, promoting the permeability of intermediate metabolic membranes, and ultimately producing specific physiological effects by controlling DNA transcription or influencing specific protein synthesis. Therefore, peptides are crucial to human cellular activity and function. However, due to various factors, the modern human body loses and depletes peptides, greatly weakening the ability to synthesize them. Therefore, the modern human body is deficient in peptides and must supplement with synthetic peptides.

[0003] Bis(4-(octadecyloxy)phenyl)methanol (TAGC) is a liquid carrier material primarily used in peptide synthesis and serves as a key starting material. The quality of TAGC directly impacts the quality of the resulting peptides, necessitating rigorous quality testing and synthesis monitoring. However, existing technologies do not disclose methods for detecting TAGC. Summary of the Invention

[0004] In view of this, an object of the present invention is to provide a method for detecting bis(4-(octadecyloxy)phenyl)methanol, which can accurately detect bis(4-(octadecyloxy)phenyl)methanol.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] The invention provides a method for detecting bis(4-(octadecyloxy)phenyl)methanol (TAGC). Liquid chromatography is used to detect products containing TAGC, wherein the mobile phase comprises a tetrahydrofuran-acetonitrile mixed solution and a formic acid aqueous solution.

[0007] Preferably, gradient elution is used in the detection process.

[0008] Preferably, the gradient elution procedure is as follows:

[0009]

[0010] Preferably, the volume ratio of tetrahydrofuran to acetonitrile in the tetrahydrofuran-acetonitrile mixed solution is 3 to 5:1.

[0011] Preferably, the volume concentration of formic acid in the formic acid aqueous solution is 0.1 to 0.3%.

[0012] Preferably, in the detection process, the stationary phase of the chromatographic column used is C18, C8, C16 or C30.

[0013] Preferably, during the detection process, the column temperature of the chromatographic column used is 25-35°C.

[0014] Preferably, the detection process further includes: pre-treating the product containing TAGC.

[0015] Preferably, the pretreatment is to dilute the TAGC-containing product with a diluent.

[0016] Preferably, the flow rate of the mobile phase is 0.8 to 1.2 mL / min.

[0017] Preferably, the detection uses an ultraviolet detector with a detection wavelength of 230 to 240 nm.

[0018] The present invention provides a method for detecting TAGC. Using a tetrahydrofuran-acetonitrile mixture and aqueous formic acid as the mobile phase, liquid chromatography is used to detect products containing TAGC, providing accurate detection results. Experimental results demonstrate that the method for detecting TAGC provided by the present invention has good system applicability, strong specificity, good precision, and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The chromatogram is a TAGC detection using tetrahydrofuran-acetonitrile as the organic phase;

[0020] Figure 2 The chromatogram of the crude TAGC product was detected using tetrahydrofuran-acetonitrile as the organic phase;

[0021] Figure 3 The chromatogram is for detecting TAGC using acetonitrile as the organic phase;

[0022] Figure 4 The chromatogram of TAGC was detected using tetrahydrofuran as the organic phase;

[0023] Figure 5 Specific detection spectrum of the TAGC detection method provided by the present invention;

[0024] Figure 6 This is a sensitivity detection graph of the TAGC detection method provided by the present invention. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] The invention provides a method for detecting bis(4-(octadecyloxy)phenyl)methanol (TAGC). Liquid chromatography is used to detect products containing TAGC, wherein the mobile phase comprises a tetrahydrofuran-acetonitrile mixed solution and a formic acid aqueous solution.

[0027] TAGC is a liquid-phase carrier material prepared using liquid-phase synthesis of carbon-based tags. The presence of long aliphatic chains significantly increases the hydrophobicity of the peptide-tag, and this high hydrophobicity is not affected by peptide chain growth. These carbon-based tags can be precipitated in polar solvents such as acetonitrile or methanol, providing a foundation for the separation and extraction of intermediates in amino acid condensation and Fmoc deprotection. The TAGC-containing products described herein can be products, intermediates, or raw materials from the TAGC synthesis process. The present invention does not specifically limit the source of the TAGC-containing products.

[0028] A TAGC-containing product is weighed as a test sample. In some possible implementations, the test sample is diluted with a diluent, preferably a tetrahydrofuran-dichloromethane mixed solvent. After the diluent is added, ultrasonic treatment is performed for 1 to 5 minutes, preferably 1 to 2 minutes. After the ultrasonication is completed, the volume is fixed with the diluent and shaken to obtain a test sample solution. In some possible implementations, the test sample solution is filtered, preferably using an organic phase filter membrane with a specification of 0.22 to 0.65 μm, preferably 0.45 μm, to obtain a filtrate of the test sample.

[0029] After filtration, the filtrate of the sample to be tested is subjected to liquid chromatography detection. The liquid chromatography detection conditions are as follows:

[0030] In the above-mentioned liquid chromatography detection, the stationary phase of the chromatographic column used is C18, C8, C16 or C30, preferably C18. The specifications of the chromatographic column can be 4.6×250mm×5μm, 2.1×250mm×5μm or 10×250mm×5μm, preferably 4.6×250mm×5μm. The column temperature of the chromatographic column is 25-35°C, preferably 30°C. The present invention has no special restrictions on the source of the chromatographic column and its stationary phase, and they can be purchased on the market.

[0031] The mobile phase includes an organic phase and an aqueous phase. The organic phase is a tetrahydrofuran-acetonitrile mixed solution, the volume ratio of tetrahydrofuran:acetonitrile is 3-5:1, preferably 4:1, the aqueous phase is a formic acid aqueous solution, the volume concentration of formic acid is 0.1-0.3%, preferably 0.1%, and the flow rate of the mobile phase is 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0032] In the liquid chromatography detection, gradient elution is adopted, and the gradient elution procedure is as follows:

[0033]

[0034] In the liquid chromatography detection, the injection volume is 3 to 8 μL, preferably 5 μL. The detector used in the liquid chromatography detection is an ultraviolet detector, and the detection wavelength is 230 to 240 nm, preferably 235 nm.

[0035] The filtrate of the above-mentioned sample to be tested is tested using the above-mentioned chromatographic detection conditions, and the purity of TAGC in the sample to be tested is calculated by the area normalization method.

[0036] The present invention also conducts a methodological investigation on the detection method of TAGC provided, including system suitability investigation, specificity investigation, precision investigation and sensitivity investigation.

[0037] The present invention provides a method for detecting TAGC, using a tetrahydrofuran-acetonitrile mixed solution and an aqueous formic acid solution as the mobile phase. Using the tetrahydrofuran-acetonitrile mixed solution as the organic phase increases the mobile phase's elution capacity for TAGC, resulting in accurate detection results. Furthermore, a methodological investigation of the TAGC detection method provided by the present invention was conducted, and experimental results demonstrated that the method provided by the present invention exhibits good system applicability, strong specificity, good precision, and high sensitivity.

[0038] Example 1:

[0039] (1) Sample preparation

[0040] The product of the TAGC synthesis process using the carbon-based tag liquid-phase synthesis method was used as the test sample. Accurately weigh 25 mg of the sample and place it in a 25 mL volumetric flask. Add an appropriate amount of diluent (tetrahydrofuran-dichloromethane mixed solvent) to dilute the sample. Ultrasonicate and dissolve for 1-2 minutes. Dose the diluent to volume and shake well. Filter an appropriate amount of the sample solution through a 0.45 μm organic phase filter to obtain the sample filtrate for testing.

[0041] (2) Liquid chromatography detection conditions

[0042] The chromatographic column used was Thermo hypersil GOLD (4.6×250 mm×5 μm), the mobile phase was 80% tetrahydrofuran-acetonitrile solution and 0.1% formic acid aqueous solution, and the gradient elution program was:

[0043]

[0044] The flow rate of the mobile phase was 1.0 mL / min, the column temperature was 30 °C, the detection wavelength was 235 nm, and the injection volume was 5.0 μL.

[0045] (3) Sample testing

[0046] The chromatographic detection conditions described in step (2) are used to detect the sample filtrate obtained in step (1) to obtain the following Figure 1 The chromatogram shown, Figure 1 The chromatographic peaks and their elution times are shown in Table 1. Figure 1 As shown in Table 1, the mobile phase using a tetrahydrofuran-acetonitrile mixed solution as the organic phase can successfully elute TAGC. The purity of TAGC contained in the sample to be tested was calculated using the area normalization method to be 97.0%.

[0047] Table 1

[0048]

[0049] Example 2

[0050] (1) Sample preparation

[0051] The intermediate product from the carbon-based tag liquid-phase synthesis process was used as the sample. Accurately weigh 25 mg of the sample and place it in a 25 mL volumetric flask. Add an appropriate amount of diluent (tetrahydrofuran-dichloromethane mixed solvent) to disperse the sample. Dissolve the sample by ultrasonication for 1-2 minutes, then dilute to volume with the diluent and shake well. An appropriate amount of the sample solution was filtered through a 0.45 μm organic phase filter to obtain the sample filtrate for testing.

[0052] (2) Instrumental chromatographic conditions

[0053] The chromatographic column used was Thermo hypersil GOLD (4.6×250 mm×5 μm), the mobile phase was 80% tetrahydrofuran-acetonitrile solution and 0.1% formic acid-water system, and the gradient elution program was:

[0054]

[0055] The flow rate of the mobile phase was 1.0 mL / min, the column temperature was 30 °C, the detection wavelength was 235 nm, and the injection volume was 5.0 μL.

[0056] (3) Sample testing

[0057] The chromatographic detection conditions described in step (2) are used to detect the sample filtrate obtained in step (1) to obtain the following Figure 2 The chromatogram shown, Figure 2 The chromatographic peaks and their elution times are shown in Table 2. Figure 2 As shown in Table 2, using a tetrahydrofuran-acetonitrile mixed solution as the organic phase, TAGC and C1 (intermediate synthesis product) can be baseline separated. The purity of TAGC contained in the test sample was calculated to be 88.6% by area normalization method.

[0058] Table 2

[0059]

[0060] Comparative Example 1

[0061] The test sample in Example 1 was tested by the detection method of Example 1, but the organic phase was changed from tetrahydrofuran-acetonitrile mixed solution to acetonitrile. The test results are shown in FIG. Figure 3 ,Depend on Figure 3 It can be seen that when acetonitrile is used as the organic phase, the TAGC product cannot be eluted.

[0062] Comparative Example 2

[0063] The test sample in Example 1 was tested by the detection method of Example 1, but the organic phase was changed from tetrahydrofuran-acetonitrile mixed solution to tetrahydrofuran. The test results are shown in FIG. Figure 4 ,Depend on Figure 4 It can be seen that when tetrahydrofuran is used as the organic phase, TAGC and C2 (intermediate synthesis product) cannot be separated.

[0064] Example 3

[0065] The methodological investigation of the liquid chromatography detection method of TAGC provided by the present invention was carried out.

[0066] (1) System applicability investigation

[0067] The same batch of TAGC products synthesized using the carbon-based tag liquid-phase synthesis method was used as the test sample. One test solution was prepared and six injections were performed using the liquid chromatography detection method provided in Example 1. The calculated results are shown in Table 3. As shown in Table 3, the relative standard deviation of the peak area detection results is small, indicating that the TAGC detection method provided by the present invention has good system applicability.

[0068] Table 3

[0069]

[0070] (2) Precision inspection

[0071] Six sample solutions of TAGC synthesized using the same batch of carbon-based tag liquid-phase synthesis were prepared. Each sample was injected once using the detection method described in Example 1. The calculated results are shown in Table 4. As shown in Table 4, the relative standard deviation of the purity test results is small, indicating that the detection method for TAGC provided by the present invention has good precision.

[0072] Table 4

[0073]

[0074] (3) Specificity inspection

[0075] A mixed solution of products and intermediates in the process of synthesizing TAGC by carbon-based tag liquid phase synthesis was prepared and analyzed by the detection method provided in Example 1. The separation degree was greater than 2.0, and the chromatogram was as follows: Figure 5 As shown, Figure 5 The chromatographic peaks and their elution times are shown in Table 5. Figure 5 As shown in Table 5, TAGC and C2 can achieve baseline separation, indicating that the detection method of TAGC provided by the present invention has strong specificity.

[0076] Table 5

[0077]

[0078] (4) Sensitivity investigation

[0079] Using the stepwise dilution method and the detection method provided in Example 1, TAGC solutions of different concentrations were analyzed. When the signal-to-noise ratio was not less than 10, the lowest quantitative detection concentration was only 0.1 mg / g, indicating that the detection method of TAGC provided by the present invention has good sensitivity. Figure 6 shown.

[0080] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting bis(4-(octadecyloxy)phenyl)methanol, characterized in that: Liquid chromatography was used to detect products containing bis(4-(octadecyloxy)phenyl)methanol, and the mobile phase consisted of a tetrahydrofuran-acetonitrile mixed solution and a formic acid aqueous solution. Gradient elution was used during the detection process; The gradient elution procedure is as follows:

2. The detection method according to claim 1, wherein The volume ratio of tetrahydrofuran to acetonitrile in the tetrahydrofuran-acetonitrile mixed solution is 3 to 5:

1.

3. The detection method according to claim 1, wherein The volume concentration of formic acid in the formic acid aqueous solution is 0.1-0.3%.

4. The detection method according to claim 1, wherein In the detection process, the stationary phase of the chromatographic column used is C18, C8, C16 or C30.

5. The detection method according to claim 1, wherein During the detection process, the column temperature of the chromatographic column used is 25-35°C.

6. The detection method according to claim 1, characterized in that The detection process also includes: pre-treating the product containing bis(4-(octadecyloxy)phenyl)methanol.

7. The detection method according to claim 6, characterized in that The pretreatment is to dilute the product containing bis(4-(octadecyloxy)phenyl)methanol with a diluent.

8. The detection method according to claim 1, wherein The flow rate of the mobile phase is 0.8-1.2 mL / min.

9. The detection method according to claim 1, wherein The detection is performed using an ultraviolet detector with a detection wavelength of 230 to 240 nm.

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