Method for determining eight active components in dendrobium or cosmetics

Abstract

The invention discloses a method for determining eight active components in dendrobium or cosmetics, which comprises the following steps: (1) pretreating a sample to obtain a solution for instrumental analysis; (2) detecting the eight active components in the sample by adopting a liquid chromatography-mass spectrometry method; the eight active ingredients are quercetin, naringenin, rutin, apigenin, caffeic acid, ferulic acid, syringic acid and chlorogenic acid. According to the method disclosed by the invention, extraction, chromatographic separation and mass spectrum quantification and qualitation of eight active substances including quercetin, naringenin, rutin, apigenin, caffeic acid, ferulic acid, syringic acid and chlorogenic acid in the dendrobe and the cosmetics are realized, and a technical support and a scientific basis are provided for research on efficacy relevance of active components of the dendrobe in the cosmetics.

Description

Technical Field

[0001] The invention relates to a method for detecting chemical substances, in particular to a method for determining eight active ingredients in dendrobium or cosmetics. Background Art

[0002] As a natural plant and traditional precious Chinese medicinal material, Dendrobium contains a variety of ingredients such as polysaccharides, stilbenes, alkaloids, amino acids and minerals. Its antioxidant, anti-aging, lipid-lowering and blood sugar-lowering, and immune-enhancing effects are also well known to the public. The eight active substances, quercetin, naringenin, rutin, apigenin, caffeic acid, ferulic acid, syringic acid and chlorogenic acid, have antibacterial, antioxidant, anti-glycation, anti-aging, whitening and anti-inflammatory effects. There is no method in the prior art that can accurately detect the above eight active substances at the same time. Determination of the effective ingredients in Dendrobium is very important for promoting the application of Dendrobium in the field of cosmetics.

[0003] Sample pretreatment is a key link in the entire analysis process and directly affects the sensitivity and precision of the analysis method. The existing methods are cumbersome, time-consuming, complex and costly. It is necessary to develop appropriate sample pretreatment technology to eliminate sample matrix interference and improve the accuracy of test results. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for determining eight active ingredients in dendrobium or cosmetics with low cost and simple operation.

[0005] A method for determining eight active ingredients in dendrobium or cosmetics, comprising the following steps:

[0006] (1) Pre-treating the sample to obtain a filtrate to be tested;

[0007] (2) Liquid chromatography-mass spectrometry was used to detect eight active ingredients in the sample; the eight active ingredients were quercetin, naringenin, rutin, apigenin, caffeic acid, ferulic acid, syringic acid and chlorogenic acid.

[0008] The method for determining 8 active ingredients in dendrobium or cosmetics of the present invention, wherein the chromatographic conditions are as follows: chromatographic column: SB-C18 chromatographic column, 4.6×50mm 1.8μm; injection volume: 2μL; flow rate: 0.3min / mL; fluidity: A is 0.1% formic acid water, B is acetonitrile; gradient elution, 0.1min, A:B=90:10; 2min, A:B=50:50; 4min, A:B=10:90; 6min, A:B=10:90; 6.5min, A:B=2:98; 8min stop.

[0009] The method for determining 8 active ingredients in dendrobium or cosmetics described in the present invention comprises the following steps: electrospray ion source positive and negative ion modes, multiple reaction monitoring; capillary voltage 1.5 kV; cone gas flow: nitrogen, flow rate 50 L / h; desolvation temperature 500° C., flow rate 800 L / h; ion source temperature 150° C.; nebulizing gas pressure 0.28 MPa; collision gas: high-purity argon; mass spectrometry parameters are optimized using a single standard solution of each phenolic compound at 500.0 μg / L to determine characteristic parent ions and daughter ions.

[0010] The method for determining 8 active ingredients in dendrobium or cosmetics described in the present invention, wherein: mass spectrometry detection retention time, parent ion, daughter ion and CE voltage are shown in Table 1.

[0011] Table 1 Retention time, parent ion, daughter ion and CE voltage of each analyte

[0012]

[0013] The method for determining 8 active ingredients in dendrobium or cosmetics described in the present invention, wherein the sample is a cosmetic sample of three matrices of liquid, cream or mask; the sample pretreatment method is as follows: accurately weigh 1.0g of sample in a 10mL stoppered colorimetric tube, add methanol to the scale, vortex mix, perform ultrasound-assisted extraction for 20min, vortex mix and let stand, take the supernatant, filter through a 0.22μm organic filter membrane, and the filtrate is to be tested.

[0014] The method for determining eight active ingredients in dendrobium or cosmetics of the present invention, wherein the sample is dendrobium; the sample pretreatment method is as follows: accurately weigh 0.25g of ground dendrobium powder and place it in a 25mL colorimetric tube, add 10mL of 70% methanol aqueous solution, place it in a constant temperature oscillator at 50°C water bath for 30min, cool it naturally to room temperature, transfer the extract to a centrifuge tube, and swirl it at 8000r·min -1 Centrifuge for 6 minutes and transfer the supernatant to a 25mL volumetric flask; extract the sample again with 70% methanol aqueous solution, combine the extracts, make up to 25mL, shake well, filter with 0.22μm filter membrane and then test. If the detected concentration of the component in the test solution exceeds the linear range of the standard curve, it can be appropriately diluted with 70% methanol aqueous solution and then analyzed.

[0015] The method for determining 8 active ingredients in dendrobium or cosmetics of the present invention further comprises the preparation of standard solutions: accurately weighing 10.0 mg of each of the 8 compounds, dissolving them in methanol to a volume of 10.00 mL, obtaining a single-standard mother solution with a concentration of 1000 mg / L for each compound, diluting the mother solution to prepare a 10.0 mg / L single-standard intermediate solution, storing the solution in a -20°C refrigerator, and using the mixed standard solutions of different concentrations for preparing a standard working curve, which are prepared and used immediately.

[0016] The method for determining eight active ingredients in dendrobium or cosmetics of the present invention is different from the prior art in that:

[0017] The detection method of the present invention optimizes the extraction conditions of dendrobium and cosmetics, and uses liquid chromatography-mass spectrometry to determine the extraction of quercetin, naringenin, rutin, apigenin, caffeic acid, ferulic acid, syringic acid and chlorogenic acid 8 active substances with antibacterial, antioxidant, anti-glycation, anti-aging, whitening and anti-inflammatory effects from dendrobium. The results show that these 8 substances have good separation and better qualitative effect under the SB-C18 chromatographic column (4.6×50mm 1.8μm) using gradient elution conditions. In quantitative analysis, these 8 substances have good linear relationships within their respective ranges, the correlation coefficients are all greater than 0.999, and the detection limits are 0.03, 0.03, 0.03, 0.03, 0.15, 0.03, 0.03, 0.15mg / kg, respectively.

[0018] The method of the invention realizes the separation, extraction and determination of 8 active substances including quercetin, naringenin, rutin, apigenin, caffeic acid, ferulic acid, syringic acid and chlorogenic acid in dendrobium and cosmetics, and provides a theoretical basis for the research on the antibacterial, antioxidant, anti-glycation, anti-aging, whitening and anti-inflammatory effects of dendrobium in cosmetics.

[0019] The method for determining eight active ingredients in cosmetics of the present invention will be further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 chromatograms of 8 substances in the method of the present invention;

[0021] Figure 2 Graph showing the recovery rates of different extraction solvents in the method of the present invention;

[0022] Figure 3 It is the recovery rate diagram of different extraction times in the method of the present invention. DETAILED DESCRIPTION

[0023] 1 Materials and methods

[0024] 1.1 Instruments and reagents

[0025] 1.1.1. Instruments and equipment

[0026] AB SCIEX QTRAP 5500 high performance liquid chromatography tandem mass spectrometer, AB SCIEX, USA; MX-S vortex oscillator (SCILOGEX, USA); XSE204 analytical balance (Mettler-Toledo, USA); Milli-Q ultrapure water generator (Millipore, USA).

[0027] 1.1.2 Reagents and standards

[0028] Acetonitrile, methanol (chromatographic grade, Thermo Company, USA); formic acid (chromatographic grade, Merck, USA). Quercetin (purity 94.5%), naringenin (purity 99.0%), rutin (purity 95.6%), apigenin (purity 95.9%), caffeic acid (purity 99.0%), ferulic acid (purity 99.6%), syringic acid (purity 98.0), chlorogenic acid (purity 97.5%) were all purchased from Shanghai Anpu Laboratory Technology Co., Ltd. Dendrobium from different origins, purchased online.

[0029] 1.2 Test conditions

[0030] Chromatographic column: SB-C18 chromatographic column (4.6×50mm 1.8μm); injection volume: 2μL; flow rate: 0.3min / mL; fluidity: A is 0.1% formic acid water, B is acetonitrile; gradient elution, 0.1min (A:B=90:10), 2min (A:B=50:50), 4min (A:B=10:90), 6min (A:B=10:90), 6.5min (A:B=2:98), 8min stop.

[0031] Mass spectrometry conditions: electrospray ion source positive and negative ion modes, multiple reaction monitoring; capillary voltage 1.5 kV; cone gas flow: nitrogen, flow rate 50 L / h; desolvation temperature 500 ° C, flow rate 800 L / h; ion source temperature 150 ° C; nebulizer gas pressure 0.28 MPa; collision gas: high-purity argon. The mass spectrometry parameters were optimized with a single standard solution of each phenolic compound at 500.0 μg / L to determine the characteristic parent ions and daughter ions. The specific parameters of the mass spectrometry detection of qualitative and quantitative ion pairs and the optimized cone voltage and collision energy are shown in Table 1.

[0032] Table 1 Retention time, parent ion, daughter ion and CE voltage of each analyte

[0033]

[0034] 1.3 Preparation of standard solution

[0035] Accurately weigh 10.0 mg of each of the eight compounds, dissolve them in methanol to 10.00 mL, and obtain a single standard stock solution with a concentration of 1000 mg / L for each compound. Dilute the stock solution to prepare a 10.0 mg / L single standard intermediate solution, and store it in a -20°C refrigerator. Mixed standard solutions of different concentrations used to prepare standard working curves should be prepared and used immediately.

[0036] 1.4 Sample pretreatment

[0037] 1.4.1 Extract of Dendrobium officinale: 0.25 g of ground Dendrobium officinale powder was accurately weighed and placed in a 25 mL colorimetric tube. 10 mL of 70% methanol aqueous solution was added and placed in a constant temperature oscillator at 50 °C for 30 min. The extract was naturally cooled to room temperature and transferred to a centrifuge tube. The extract was centrifuged at 8000 r / min. -1 Centrifuge for 6 minutes, transfer the supernatant to a 25 mL volumetric flask. Repeat the extraction of the sample with 70% methanol aqueous solution, combine the extracts, make up to 25 mL, shake well, filter with a 0.22 μm filter membrane and test. If the concentration of the components in the test solution exceeds the linear range of the standard curve, dilute it appropriately with 70% methanol aqueous solution and then analyze it.

[0038] 1.4.2 Sample: Accurately weigh 1.0 g of sample into a 10 mL stoppered colorimetric tube, add methanol to the scale, vortex mix, perform ultrasonic-assisted extraction for 20 min, vortex mix and let stand, take the supernatant, filter through a 0.22 μm organic filter membrane, and the filtrate is tested.

[0039] 2 Results

[0040] 2.1 Mass spectra of each substance to be tested

[0041] The response and ionization degree of methanol and acetonitrile as organic mobile phases to the compounds were investigated respectively. The results showed that 0.1% formic acid water-acetonitrile had the highest response and the best peak shape for each compound. At the same time, the gradient elution program of the mobile phase system was optimized to achieve the best test effect for each compound.

[0042] The substances to be tested were determined according to the method in the test conditions. The mass spectrometry detection parameters under the optimized conditions are shown in Table 1. The chromatograms of the compounds are shown in Figure 1 .

[0043] 2.2 Methodological validation of the detection of eight compounds

[0044] According to the response of the target compound ion, mixed standard solutions of different concentrations were prepared, and the samples were injected under the optimized conditions. The standard curve was established with the standard solution concentration (x, μg / kg) and the corresponding peak area (y) to obtain the linear regression equation.

[0045] As shown in Table 2, the eight substances showed good linear relationships within their respective linear ranges, with correlation coefficients greater than 0.999. The detection limits and quantification limits of the eight test compounds were calculated using 3-fold and 10-fold signal-to-noise ratios, respectively, and the detection limits were between 0.03 and 0.15 mg / kg, and the quantification limits were between 0.1 and 0.5 mg / kg.

[0046] Table 2 Linear range and correlation coefficients of 8 compounds

[0047]

[0048] 2.3 Spike recovery experiments of eight compounds in different sample matrices

[0049] Recovery rate and relative standard deviation The cosmetic samples without the analyte were used as blank matrix, and mixed standard solutions of low, medium and high levels were added respectively. The determination was carried out according to this method. Each matrix and each level were measured in parallel 6 times, and the spiked recovery rate was calculated. The results showed that the recovery rate of liquid matrix samples was 88.6% to 114.9%, with a relative standard deviation of 1.9% to 8.3%, the recovery rate of cream matrix samples was 88.2% to 117.6%, with a relative standard deviation of 2% to 8.6%, and the recovery rate of mask matrix samples was 88.7% to 118.4%, with a relative standard deviation of 2.1% to 8.3%. All three matrix samples can meet the actual detection needs.

[0050] Table 3 Recovery and precision of samples (n=6)

[0051]

[0052] 2.4 Selection of sample extraction solvent

[0053] Three cosmetic samples in liquid, cream and mask matrix were selected, and acetonitrile, methanol and chloroform were selected to extract 8 compounds in the samples under the same method (1.4.2). The results showed that the recovery rate of acetonitrile was 86..3% to 92.9%, with a relative standard deviation of 2.3% to 3.3%, the recovery rate of methanol was 90.6% to 98.4%, with a relative standard deviation of 1.2% to 2.6%, and the recovery rate of chloroform was 83.5% to 88.7%, with a relative standard deviation of 1.1% to 2.8%. Therefore, methanol has a higher recovery rate, and methanol is selected as the extraction solvent. See Table 4. Figure 2 .

[0054] Table 4 Recovery and RSD of different extraction solvents

[0055]

[0056] 2.5 Selection of sample extraction time

[0057] Select three cosmetic samples with three matrices: liquid, cream and mask. Select different extraction times of 5, 10, 15, 20, 25 and 30 minutes. Use methanol to extract 8 compounds in the samples under the same operation mode (1.4.2). The results show that the optimal extraction time is 20 minutes, the recovery rate of liquid matrix is ​​99.9%, the recovery rate of cream matrix is ​​101.2%, and the recovery rate of mask matrix is ​​98.9%, see Table 5 and Figure 3 .

[0058] Table 5 Recovery and RSD of different extraction times

[0059]

[0060] 2.6 Determination of content in samples

[0061] Different samples of Dendrobium officinale were selected, processed according to the method of "1.4.1", and tested on the machine. The test results are shown in Table 6 ("ND" means not detected).

[0062] Table 6 Detection results of the contents of 8 active ingredients in the Dendrobium officinale extract samples (mg / kg)

[0063]

[0064] Select three cosmetic samples of liquid, cream and mask matrix, process them according to the method of "1.4.2", and test them on the machine. The test results are shown in Table 7 ("ND" means not detected).

[0065] Table 7 Test results of 8 active ingredients in cosmetic samples (mg / kg)

[0066] Target Sample 1 (liquid milk) Sample 2 (cream) Sample 3 (Facial Mask) Quercetin ND ND ND Naringenin 0.12 0.17 0.11 Rutin 0.10 0.15 ND Apigenin ND ND ND Caffeic acid ND ND ND Ferulic acid 0.16 0.13 0.32 Syringic acid ND ND ND Chlorogenic acid ND ND ND

[0067] The method for determining 8 active ingredients in dendrobium or cosmetics provided by the present invention not only has good detection limits and quantification limits (the detection limits are 0.03, 0.03, 0.03, 0.03, 0.15, 0.03, 0.03 and 0.15 mg / kg respectively; the quantification limits are 0.1, 0.1, 0.1, 0.1, 0.5, 0.1, 0.1 and 0.5 mg / kg respectively), but also has good recovery rates, and the recovery rates of different matrices are between 88.6% and 117.6%, and the RSD is between 1.9% and 8.6%.

[0068] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for determining eight active ingredients in dendrobium or cosmetics, characterized in that: The steps include: (1) Pre-treating the sample to obtain a filtrate to be tested; (2) Liquid chromatography-mass spectrometry was used to detect eight active ingredients in the sample; the eight active ingredients were quercetin, naringenin, rutin, apigenin, caffeic acid, ferulic acid, syringic acid and chlorogenic acid.

2. The method for determining eight active ingredients in dendrobium or cosmetics according to claim 1, characterized in that: The chromatographic conditions are as follows: chromatographic column: SB-C18 chromatographic column, 4.6×50mm 1.8μm; injection volume: 2μL; flow rate: 0.3min / mL; fluidity: A is 0.1% formic acid water, B is acetonitrile; gradient elution, 0.1min, A:B=90:10; 2min, A:B=50:50; 4min, A:B=10:90; 6min, A:B=10:90; 6.5min, A:B=2:98; 8min stop.

3. The method for determining 8 active ingredients in dendrobium or cosmetics according to claim 2, characterized in that: The mass spectrometry conditions are as follows: electrospray ion source positive and negative ion modes, multiple reaction monitoring; capillary voltage 1.5 kV; cone gas flow: nitrogen, flow rate 50 L / h; desolvation temperature 500°C, flow rate 800 L / h; ion source temperature 150°C; nebulizer gas pressure 0.28 MPa; collision gas: high-purity argon; mass spectrometry parameters were optimized using 500.0 μg / L single standard solutions of each phenolic compound to determine the characteristic parent ions and daughter ions.

4. The method for determining eight active ingredients in dendrobium or cosmetics according to claim 3, characterized in that: Table 1 Retention time, parent ion, daughter ion and CE voltage of each analyte The retention time, parent ion, product ion and CE voltage of mass spectrometry detection are shown in Table 1.

5. The method for determining eight active ingredients in dendrobium or cosmetics according to claim 1, characterized in that: The samples are cosmetic samples of three matrices: liquid, cream or mask; the sample pretreatment method is as follows: accurately weigh 1.0 g of the sample into a 10 mL stoppered colorimetric tube, add methanol to the scale, vortex mix, perform ultrasound-assisted extraction for 20 minutes, vortex mix and let stand, take the supernatant, filter through a 0.22 μm organic filter membrane, and the filtrate is tested.

6. The method for determining eight active ingredients in dendrobium or cosmetics according to claim 1, characterized in that: The sample is dendrobium; the sample pretreatment method is as follows: accurately weigh 0.25g of ground dendrobium powder and place it in a 25mL colorimetric tube, add 10mL of 70% methanol aqueous solution, place it in a constant temperature oscillator at 50°C water bath for 30min, cool it naturally to room temperature, transfer the extract to a centrifuge tube, and simmer at 8000r·min -1 Centrifuge for 6 min and transfer the supernatant to a 25 mL volumetric flask; Repeat the extraction of the sample with 70% methanol aqueous solution, combine the extracts, make up to 25 mL, shake well, filter with 0.22 μm filter membrane and then test. If the detected concentration of the component in the test solution exceeds the linear range of the standard curve, it can be appropriately diluted with 70% methanol aqueous solution and then analyzed.

7. The method for determining eight active ingredients in cosmetics according to claim 1, characterized in that: The method also includes the preparation of standard solutions: accurately weighing 10.0 mg of each of the eight compounds, dissolving them in methanol to a volume of 10.00 mL, respectively, to obtain a single standard mother solution with a concentration of 1000 mg / L for each compound, diluting the mother solution to prepare a 10.0 mg / L single standard intermediate solution, storing it in a -20°C refrigerator, and using it to prepare mixed standard solutions of different concentrations for the standard working curve, which are prepared and used immediately.

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