A qualitative and quantitative detection method for phytosphingosine

Abstract

The present application relates to the field of biotechnology, and discloses a qualitative and quantitative detection method for phytosphingosine. The method first determines the peak area of ​​phytosphingosine in a standard solution by high performance liquid chromatography, then determines the peak area of ​​a sample by high performance liquid chromatography, and finally calculates the content of phytosphingosine in the sample by using a formula. During the high performance liquid chromatography determination process, mobile phase A and mobile phase B are used for elution, wherein the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, and the mass fraction of formic acid in the formic acid aqueous solution is 0.05-0.1%. Through the above design, the present application can separate phytosphingosine in the sample by using high performance liquid chromatography, and achieves separation and purification of phytosphingosine by selecting the mobile phase. Moreover, since the present application does not need to use mass spectrometry for further qualitative and quantitative analysis results of phytosphingosine, its operation steps are simpler and the test speed is faster.

Description

Technical Field

[0001] The invention relates to the technical field of detection and analysis, and in particular to a qualitative and quantitative detection method for phytosphingosine. Background Art

[0002] Phytosphingosine is the latest generation of moisturizer developed in recent years. It is a water-soluble lipid substance. It is similar to the material structure of the stratum corneum of the skin. It can quickly penetrate into the skin and combine with the water in the stratum corneum to form a mesh structure to lock in moisture. As we age and enter old age, the ceramide in human skin will gradually decrease. The appearance of skin abnormalities such as dry skin and rough skin is also due to the decrease in ceramide. Therefore, it is necessary to prevent such skin abnormalities.

[0003] The functions of phytosphingosine in cosmetics include: promoting the production of skin ceramides (the conversion requires a metabolic cycle of 28 days), and is the key supplement for skin barrier deficiency; relieving skin irritation and soothing inflammation; having potential antibacterial activity, which can eliminate many harmful bacteria on the skin and reduce bacterial damage to the skin; inhibiting TPA-induced epidermal growth; stimulating the differentiation of human epidermal keratinocytes, a function that can be simply understood as anti-aging.

[0004] Chinese patent application 202010156913.8 discloses a method for detecting glucosylceramide. It includes the following steps: obtaining a dried blood spot sample; adding an acetonitrile aqueous solution containing a glucosylceramide isotope internal standard to the dried blood spot sample, and shaking to obtain a sample to be tested; using liquid chromatography tandem mass spectrometry to detect the sample to be tested; the method for detecting glucosylceramide disclosed in this scheme is based on liquid chromatography tandem mass spectrometry technology, using dried blood spots as test samples, using glucosylceramide isotope internal standard as internal standard, and extracting by shaking with an acetonitrile aqueous solution, which has simple pretreatment, requires a small amount of sample, and is easy to collect, transport and store;

[0005] Further observation of the scheme shows that the scheme selects a formic acid aqueous solution with a volume concentration of 0.09% to 0.11% as the mobile phase A, and selects a formic acid acetonitrile solution with a volume concentration of 0.09% to 0.11% as the mobile phase B.

[0006] Chinese patent application 202211154911.0 discloses a method for separating and identifying phytosphingosine and / or N-acetylphytosphingosine, comprising the following steps: using high performance liquid chromatography to separate and detect a reference solution of phytosphingosine or N-acetylphytosphingosine to obtain a first chromatogram; using the high performance liquid chromatography to separate and detect a sample containing phytosphingosine and / or N-acetylphytosphingosine to obtain a second chromatogram; determining the phytosphingosine and / or N-acetylphytosphingosine and its content in the sample according to the first chromatogram, the second chromatogram and the concentration of the reference solution of phytosphingosine or N-acetylphytosphingosine; the method quickly, accurately and effectively detects the content of phytosphingosine and N-acetylphytosphingosine, providing a reliable detection and analysis method for the quality control of phytosphingosine; the method has the characteristics of simple operation, accurate and reliable data and good reproducibility;

[0007] Further observation of the scheme shows that the scheme uses trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B.

[0008] The problem that this scheme needs to solve is: how to provide a new qualitative and quantitative detection method for phytosphingosine and further simplify the detection steps. Summary of the invention

[0009] The purpose of this application is to provide a new qualitative and quantitative detection method for phytosphingosine and to further simplify the detection steps.

[0010] To achieve the above object, the present invention provides the following technical solution: a qualitative and quantitative detection method of phytosphingosine, comprising the following steps:

[0011] Step 1: Determine the peak area of ​​phytosphingosine in the standard solution by high performance liquid chromatography, and draw a standard curve with the logarithm of the injected mass as the abscissa and the logarithm of the average peak area as the ordinate;

[0012] Step 2: Determine the peak area of ​​phytosphingosine in the sample by HPLC, and calculate the log 10 m 样 ;

[0013] Step 3: Calculate the content of phytosphingosine in the sample using formula 1;

[0014]

[0015] Where: ω is the content of phytosphingosine in the sample;

[0016] m 样 is the sample injection mass, in μg;

[0017] V0 is the sample fixed volume, in mL;

[0018] F is the dilution factor;

[0019] V1 is the sample injection volume, in μL;

[0020] m1 is the sample weight, in g;

[0021] The high performance liquid chromatography method is eluted with mobile phase A and mobile phase B;

[0022] The mobile phase A is acetonitrile;

[0023] The mobile phase B is a formic acid aqueous solution, and the mass fraction of formic acid in the formic acid aqueous solution is 0.05-0.1%;

[0024] Further preferably, the mass fraction of formic acid in the formic acid solution includes but is not limited to 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, and 0.10%.

[0025] It should be noted that F is the dilution factor, which changes with the dilution multiple of the sample. When the concentration of the sample is diluted to 20%, the value of F is 0.2, and when the sample is not diluted, the value of F is 1.

[0026] Preferably, the chromatographic column used in the high performance liquid chromatography method is selected from any one of a C18 chromatographic column and a XAmide amide chromatographic column;

[0027] Further preferably, the chromatographic column used in the high performance liquid chromatography is a carbon 18 chromatographic column.

[0028] Preferably, the length of the chromatographic column used in the high performance liquid chromatography method is 100 to 250 mm, the inner diameter of the chromatographic column is 4.0 to 5.0 mm, and the filler particle size is 4.0 to 6.0 um;

[0029] Preferably, the column temperature of the chromatographic column is 30-40°C;

[0030] Further preferably, the temperature of the chromatographic column includes but is not limited to 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C.

[0031] Preferably, the gas flow rate of the ELSD detector used in the high performance liquid chromatography is 2.5 L / min;

[0032] The signal range is 2.4V;

[0033] The evaporation temperature is 50°C;

[0034] The signal gain is 3.

[0035] Preferably, the high performance liquid chromatography method uses mobile phase A and mobile phase B for gradient elution, and the gradient elution procedure is:

[0036] 0~10min, mobile phase A 40v / v%~90v / v%;

[0037] 10-20 min, mobile phase A 90 v / v%;

[0038] 20-25 min, mobile phase A 90 v / v%-40 v / v%;

[0039] 25-30 min, mobile phase A 40 v / v%;

[0040] The flow rate of the gradient elution is 0.5-1.0 mL / min;

[0041] Further preferably, the flow rate of the gradient elution includes but is not limited to 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, and 1.0 mL / min.

[0042] Preferably, the concentration of the phytosphingosine reference solution is 0.1 to 1 mg / ml;

[0043] Further preferably, the concentration of the phytosphingosine reference solution includes but is not limited to 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, and 1.0 mg / ml.

[0044] Preferably, the sample contains phytosphingosine at a concentration of 0.1 to 1 mg / ml;

[0045] Further preferably, the sample contains phytosphingosine at a concentration including but not limited to 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, and 1.0 mg / ml.

[0046] Preferably, the sample injection volume of the high performance liquid chromatography method is 1 to 10 uL;

[0047] Further preferably, the sample injection volume of the high performance liquid chromatography includes but is not limited to 1uL, 2uL, 3uL, 4uL, 5uL, 6uL, 7uL, 8uL, 9uL, and 10uL.

[0048] The beneficial effects of this application are:

[0049] The present application can separate phytosphingosine in a sample by adopting high performance liquid chromatography, and achieves separation and purification of phytosphingosine by selecting a mobile phase, and thus the technical solution of the present application realizes separation and purification of phytosphingosine, and the qualitative and quantitative detection method of phytosphingosine disclosed in the present application does not require further analysis using a mass spectrometer, and the present application can achieve qualitative and quantitative analysis of phytosphingosine only by detection by a chromatograph and calculation of the test results, and the operation steps are simpler and the test speed is faster, and, in the course of the experiment, we were pleasantly surprised to find that when mobile phase A is acetonitrile and mobile phase B is an aqueous formic acid solution, the sensitivity of impurity detection during the analysis of phytosphingosine is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The chromatogram after the standard sample is tested in Example 1;

[0051] Figure 2 This is the chromatogram after the sample to be tested is detected in Example 1;

[0052] Figure 3 This is the chromatogram of the sample to be tested after testing in Comparative Example 1;

[0053] Figure 4 This is the chromatogram of Comparative Example 2 after testing the sample to be tested. DETAILED DESCRIPTION

[0054] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0055] The present invention will be described clearly and completely below in conjunction with the embodiments of the present application. 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 creative work are within the scope of protection of the present invention.

[0056] Example 1

[0057] Step 1: Determine the peak area of ​​the standard solution of phytosphingosine by high performance liquid chromatography, and draw a standard curve (such as Figure 1 shown).

[0058] (It should be noted that in the process of drawing the standard curve, 2 μL, 5 μL, 10 μL, and 20 μL of the phytosphingosine standard solution were injected respectively for liquid chromatography analysis, and the logarithm of the injected mass (m) was used as the abscissa, and the logarithm of the average value of the peak area (A) was used as the ordinate. Combined with the regression equation: lgA=al gm+b, the standard curve was drawn)

[0059] During the experiment, the HPLC procedure and settings were as follows:

[0060] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatograph; evaporative light scattering detector (UM5800plus);

[0061] Chromatographic column: ECOSIL C18 AQ-plus (4.6 mm × 250 mm, 5 μm);

[0062] Flow rate: 0.5 mL / min;

[0063] Column temperature: 40°C;

[0064] Injection volume: 10 μL;

[0065] Mobile phase A: acetonitrile;

[0066] Mobile phase B: formic acid aqueous solution with a mass fraction of 0.1% formic acid;

[0067] Gas flow rate: 2.5L / min;

[0068] Evaporation temperature: 50℃;

[0069] Signal gain: 3;

[0070] Gradient elution was used, and the elution program is shown in Table 1:

[0071] Table 1

[0072]

[0073] Step 2: Determine the peak area of ​​phytosphingosine in the sample by HPLC and calculate the log 10 m 样 ;

[0074] Step 3: Calculate the content of phytosphingosine in the sample using formula 1;

[0075]

[0076] Where: ω is the content of phytosphingosine in the sample;

[0077] m 样 is the sample injection mass, in μg;

[0078] V0 is the sample fixed volume, in mL;

[0079] F is the dilution factor (1 if not diluted);

[0080] V1 is the sample injection volume, in μL;

[0081] m1 is the sample weight, in g;

[0082] Example 2

[0083] It is basically the same as Example 1, except that the flow rate is 0.8 mL / min.

[0084] Example 3

[0085] It is basically the same as Example 1, except that the flow rate is 1.0 mL / min.

[0086] Example 4

[0087] It is basically the same as Example 1, except that the evaporation temperature is 40°C.

[0088] Example 5

[0089] It is basically the same as Example 1, except that the evaporation temperature is 60°C.

[0090] Example 6

[0091] The method is basically the same as Example 1, except that a Huapu XAmide amide column is selected as the chromatographic column.

[0092] Comparative Example 1

[0093] The method is basically the same as Example 1, except that a formic acid aqueous solution with a volume concentration of 0.1% is used as the mobile phase A;

[0094] A 0.1% by volume formic acid-acetonitrile solution was used as mobile phase B.

[0095] Comparative Example 2

[0096] The method is basically the same as Example 1, except that a trifluoroacetic acid aqueous solution with a volume concentration of 0.05% is used as mobile phase A, and acetonitrile is used as mobile phase B.

[0097] Result analysis:

[0098] pass Figure 2-4It can be seen that when different mobile phases are used to test the same sample, the results obtained in Example 1 include 4 impurity peaks in addition to phytosphingosine, representing 4 impurities, while Comparative Examples 1 and 2 only detect 2 impurity peaks and 3 impurity peaks in addition to sphingosine, respectively. It can be seen that the mobile phase used in Example 1 is more sensitive to impurity detection, and it can more accurately reflect other impurities in the sample, that is, the content of phytosphingosine is closer to the actual value.

Claims

1. A qualitative and quantitative detection method for phytosphingosine, characterized in that: The following steps are involved: Step 1: Determine the peak area of ​​phytosphingosine in the standard solution by high performance liquid chromatography, and draw a standard curve with the logarithm of the injected mass as the abscissa and the logarithm of the average peak area as the ordinate; Step 2: Determine the peak area of ​​phytosphingosine in the sample by high performance liquid chromatography and calculate it using the standard curve drawn in step 1 ; Step 3: Calculate the content of phytosphingosine in the sample using formula 1; Formula 1 Where: ω is the content of phytosphingosine in the sample; m 样 is the sample injection mass, in μg; V0 is the fixed volume of the sample, in mL; F is the dilution factor; V1 is the sample injection volume, in μL; m1 is the sample weight, in g; And the high performance liquid chromatography is eluted with mobile phase A and mobile phase B; The mobile phase A is acetonitrile; The mobile phase B is a formic acid aqueous solution, and the mass fraction of formic acid in the formic acid aqueous solution is 0.05-0.1%; The gradient elution program was: 0-10min, mobile phase A 40v / v%-90v / v%, the rest is mobile phase B; 10-20 min, mobile phase A 90 v / v%, the rest is mobile phase B; 20-25 min, mobile phase A 90 v / v%-40 v / v%, the rest is mobile phase B; 25-30 min, mobile phase A 40 v / v%, the rest is mobile phase B; The flow rate of the gradient elution is 0.5-1.0 mL / min.

2. The method for qualitative and quantitative detection of phytosphingosine according to claim 1, characterized in that: The chromatographic column used in the high performance liquid chromatography method is selected from any one of a C18 chromatographic column and a XAmide amide chromatographic column.

3. The qualitative and quantitative detection method of phytosphingosine according to claim 1, characterized in that: The chromatographic column used in the high performance liquid chromatography method is a C18 chromatographic column.

4. The method for qualitative and quantitative detection of phytosphingosine according to claim 3, characterized in that: The length of the chromatographic column used in the high performance liquid chromatography method is 100 to 250 mm, the inner diameter of the chromatographic column is 4.0 to 5.0 mm, and the filler particle size is 4.0 to 6.0 um.

5. The qualitative and quantitative detection method of phytosphingosine according to claim 3, characterized in that: The column temperature of the chromatographic column is 30-40°C.

6. The method for qualitative and quantitative detection of phytosphingosine according to claim 3, characterized in that: The gas flow rate of the ELSD detector used in HPLC was 2.5 L / min; The signal range is 2.4V; The evaporation temperature is 50°C; The signal gain is 3.

7. The method for qualitative and quantitative detection of phytosphingosine according to claim 1, characterized in that: The concentration of the phytosphingosine reference solution is 0.1-1 mg / ml.

8. The method for qualitative and quantitative detection of phytosphingosine according to claim 1, characterized in that: The sample contains phytosphingosine at a concentration of 0.1-1 mg / ml.

9. The method for qualitative and quantitative detection of phytosphingosine according to claim 1, characterized in that: The sample injection volume of the high performance liquid chromatography method is 1 to 10 uL.

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