CN108414665B - Method for measuring gingerol content in ginger medicinal material and preparation thereof - Google Patents

Method for measuring gingerol content in ginger medicinal material and preparation thereof Download PDF

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CN108414665B
CN108414665B CN201810366956.1A CN201810366956A CN108414665B CN 108414665 B CN108414665 B CN 108414665B CN 201810366956 A CN201810366956 A CN 201810366956A CN 108414665 B CN108414665 B CN 108414665B
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魏悦
李智宁
张丽先
宁二娟
陈欣
全彦涛
李晓
范毅
马艳妮
王学方
李飞飞
陈飞
徐如冰
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Henan Napu Biotechnology Co ltd
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Henan Cogo Testing International Co ltd
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Abstract

The invention discloses a method for measuring gingerol content in ginger medicinal material and its preparation, which comprises the steps of connecting an ultra-high performance liquid chromatography with a triple quadrupole mass spectrometry detector and an ultraviolet detector, using a multi-evaluation method, using a pure N-vanillyl nonanamide reference substance which does not exist in a sample and has stable performance and is easy to obtain as an internal reference substance, establishing relative correction factors between the components and 4 gingerol components in the sample, and measuring the content of the 4 gingerol components in the ginger medicinal material and its preparation by calculating the correction factors. The method has the advantages of simple operation, high sensitivity, accuracy, high efficiency and low cost, can objectively and accurately evaluate the quality of the ginger medicinal material and the preparation thereof, is used for quality control, can solve the problem that the quality of the medicinal material and the preparation thereof cannot be objectively and reasonably controlled due to the lack of a reference substance, and has important significance for controlling the quality and ensuring the curative effect.

Description

Method for measuring gingerol content in ginger medicinal material and preparation thereof
Technical Field
The invention particularly relates to a ginger medicinal material and a method for measuring the content of gingerol in a preparation thereof.
Background
Ginger (Zingiber officinale Rosc) is also called ginger, dried ginger, white ginger and yunnan ginger, belongs to the perennial herb of Zingiberaceae (Zingifera) and is mainly distributed in the areas from southwest to southeast in China. The ginger component is multiple and complex, has unique aromatic flavor and spicy taste, and the unique sensory quality mainly comes from two types of effective components: volatile ginger essential oil and ginger capsaicin without volatility, wherein the ginger essential oil provides unique aromatic flavor for the ginger; gingerol provides unique spicy taste to ginger. The ginger in different producing areas has certain difference in medicine property and efficacy due to different growing environments, researches show that the ginger has unique medicinal and edible value, and the ginger is a good Chinese medicine product and mainly used for treating wind-cold type common cold, cough and asthma, vomiting, phlegm and retained fluid, fullness, diarrhea and the like according to the traditional Chinese medicine and pharmacology. It is recorded in Shen nong Ben Cao Jing (Shen nong's herbal Jing), pungent and warm in flavor, entering spleen, stomach, kidney, heart and lung meridians, and has the effects of warming spleen and stomach for dispelling cold, restoring yang and dredging collaterals, eliminating dampness and eliminating phlegm, etc. Modern pharmacological studies show that ginger and dried ginger both have a plurality of pharmacological effects of resisting oxidation, inflammation, bacteria, tumors, ulcers and gastrointestinal bleeding, protecting gastric mucosa, improving local blood circulation and the like. An analytical method for gingerol in dried ginger is established by using an HPLC-UV technology, but the separation and preparation difficulty of a measured index reference substance is high, the analytical cost is high, and the analytical method is not easy to be widely applied as a quality control method of a ginger medicinal material. By the present time, the method for analyzing and evaluating ginger medicinal materials (including ginger and dried ginger) recorded in 'Chinese pharmacopoeia' of 2015 edition takes 6-gingerol as a quality control index of the dried ginger and takes 6-gingerol, 8-gingerol and 10-gingerol as a quality control index of the ginger, the indexes are few and single, the analysis time is long, the internal quality of the ginger medicinal materials and decoction pieces cannot be comprehensively reflected, and the current reference substances such as 6-gingerol, 8-gingerol and 10-gingerol are difficult to obtain and high in price, so that the practical application of multi-index component quality control is limited.
A multi-evaluation method is a new mode of multi-index quality evaluation suitable for the characteristics of traditional Chinese medicines, and utilizes the inherent functions and proportional relations among effective chemical components to only measure 1 component (a reference substance is easy to obtain) to realize the synchronous monitoring of a plurality of components (the reference substance is difficult to obtain or not), thereby overcoming the problem of the shortage of the reference substance, namely only measuring a certain representative component or components (easy to obtain, cheap, stable and effective) which do not exist in a sample and have similar performance, and simultaneously calculating the contents of other effective components to be measured.
Therefore, the development of a quality control method (one-test-multiple-evaluation method) which is simple in operation, high in detection sensitivity, low in cost, accurate and efficient and can simultaneously and quantitatively analyze multiple active ingredients has important practical significance for quality control of ginger medicinal materials and preparations thereof.
Disclosure of Invention
Based on the defects of the prior art, the invention aims to provide a method for measuring the content of gingerol in a ginger medicinal material and a preparation thereof, which adopts an ultra-high performance liquid chromatography to be connected with a triple quadrupole mass spectrum and an ultraviolet detector in series, adopts a pure substance N-vanillyl nonanamide (synthesized capsaicin) reference substance which does not exist in a sample, has similar performance, is stable and easy to obtain as an internal reference substance by a one-measurement and multi-evaluation technology, establishes relative correction factors of the component and 6-shogaol, 6-gingerol, 8-gingerol and 10-gingerol, and calculates the content of various gingerol components in the ginger medicinal material and the preparation thereof by the correction factors.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for measuring gingerol content in ginger medicinal material and preparations thereof comprises the following steps:
(1) preparation of control solutions
Precisely weighing 5 reference substances, namely N-vanillylnonanamide, 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol, respectively, dissolving and diluting with methanol to obtain single reference substance stock solutions, measuring the single reference substance stock solutions, diluting with methanol and uniformly mixing to obtain mixed reference substance standard solutions; meanwhile, the mass concentration of the preparation is respectively CoLAnd CoHIs of N-incenseStandard solution of oxalyl nonanamide, CoLLess than CoH(ii) a Freezing and storing the mixed reference substance standard solution and the N-vanillylnonanamide standard solution in a dark place;
(2) preparation of test solution
Weighing a ginger medicinal material or a ginger medicinal material preparation, adding the ginger medicinal material and the ginger medicinal material preparation into a first solvent respectively for extraction to obtain a ginger medicinal material extracting solution and a ginger medicinal material preparation extracting solution, and taking the ginger medicinal material extracting solution or the ginger medicinal material preparation extracting solution as a sample;
directly sampling a sample, adding the N-vanillyl nonane amide reference substance stock solution or the N-vanillyl nonane amide standard solution prepared in the step (1), mixing, and fixing the volume by using a second solvent to obtain a sample solution;
or diluting a sample with 50-100% methanol to obtain a sample mother solution, then sampling the sample mother solution, adding the N-vanillyl nonanamide reference substance stock solution or the N-vanillyl nonanamide standard solution prepared in the step (1), mixing, and fixing the volume with a second solvent to obtain a sample solution;
the content of the N-vanillylnonanamide in the test solution is 0.05-1.00 mu g/mL;
(3) relative correction factor foxAnd relative retention time RtRIs calculated by
Measuring by using UPLC-MS-DAD, sampling the mixed reference standard solution obtained in the step (1) of the series concentration, obtaining a TIC chromatogram and an MRM chromatogram, carrying out peak area integration, selecting N-vanillyl nonanamide as an internal reference compound, and respectively calculating the relative correction factors f of the N-vanillyl nonanamide to 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in the mixed reference standard solutionoxAnd relative retention time RtR；
The correction factor foxThe calculation formula of (2) is as follows:
Figure BDA0001637444630000021
in the formula: a. theoIs the peak area of reference substance N-vanillylnonanamide, CoIs an N-incense as an internal referenceMass concentration of the grass-based nonanamide control, AxPeak area of the reference x, CxThe mass concentration of a component to be detected as a reference substance x;
the relative retention time RtRThe calculation formula of (2) is as follows:
Figure BDA0001637444630000031
in the formula: t is tRxThe retention time, t, of the component to be tested, control xRoThe retention time of the reference substance N-vanillylnonanamide is shown;
(4) determination of active ingredients in test articles
Sampling the N-vanillyl nonanamide standard solution prepared in the step (1) and the test sample solution prepared in the step (2), determining by using UPLC-MS-DAD to obtain a TIC chromatogram and an MRM chromatogram, and calculating the mass concentrations of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in the test sample solution according to a formula;
the above formula is:
C'x＝(CxL'+CxH')/2
Figure BDA0001637444630000032
Figure BDA0001637444630000033
Figure BDA0001637444630000034
in the formula: cx' is the mass concentration of the component x to be determined in the test solution, CxL' is a component x to be measured in a test solution at a concentration CoLCalculated mass concentration of N-vanillylnonanamide standard solution, CxH' is a component x to be measured in a test solution at a concentration CoHThe calculated mass concentration of the N-vanillylnonanamide standard solution,Ax' is the peak area of the component x to be measured in the test solution, AoLIs CoLPeak area of N-Vanillylnonanoamide concentration standard solution, AoHIs CoHPeak area of N-vanillylnonanamide standard solution of concentration, foxThe relative correction factor of the N-vanillylnonanamide obtained in the step (3) on the tested components is shown, and N is a concentration influence factor.
Preferably, the conditions for UPLC measurement in step (3) and step (4) are: the chromatographic column is a reversed phase C18 chromatographic column, the mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is aqueous solution of formic acid with volume concentration of 0.02-0.2% or aqueous solution of acetic acid with volume concentration of 0.02-0.2%, acetonitrile is used as the mobile phase B, gradient elution is carried out, the flow rate is 0.4-0.6 mL/min, and the detection wavelength is 280nm +/-2 nm; the column temperature is 35 ℃, and the sample injection amount is 2.0-10.0 mu L.
Further, the procedure of gradient elution is:
Figure BDA0001637444630000041
more preferably, the chromatographic column is an Eclipse Plus C18 chromatographic column (100X 4.6mm,3.5 μm); the mobile phase consists of a mobile phase A and a mobile phase B, an aqueous solution of formic acid with the volume concentration of 0.1% is used as the mobile phase A, acetonitrile is used as the mobile phase B, and the flow rate is 0.5 mL/min.
Preferably, the second solvent in the step (2) is formed by uniformly mixing the mobile phase A and the mobile phase B according to the initial ratio of gradient elution.
Preferably, the conditions for MS measurement in step (3) and step (4) are: the ion source is ESI, the detection mode is MRM multi-reaction monitoring, the scanning mode is a positive ion mode, the pressure of atomizing gas is 275.8-379.2 kPa, the temperature of drying gas is 350 ℃, the flow rate of drying gas is 10-12L/min, and the voltage of a capillary is 4.0 kV.
Preferably, the condition parameters measured by the MS in step (3) and step (4) include:
the ion pairs of N-vanillyl nonanamide, 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol for qualitative and quantitative analysis, which are detected by adopting an MRM mode, have the following retention time, collision energy and fragmentation voltage:
Figure BDA0001637444630000042
wherein the MS assay conditions further comprise: ion pairs for quantitative analysis: n-vanillylnonanamide 294.1 → 137.0, 6-gingerol 277.1 → 177.1, 8-gingerol 305.1 → 177.1, 10-gingerol 333.2 → 177.1, 6-shogaol 277.1 → 137.0. Ion pairs for qualitative analysis: n-vanillylnonanamide 294.1 → 122.0, 6-gingerol 277.1 → 145.0, 8-gingerol 305.1 → 117.1, 10-gingerol 333.2 → 145.0, 6-shogaol 277.1 → 94.1.
Preferably, the first solvent in step (2) is one of absolute ethanol, acetone, methanol and water.
Preferably, the ginger medicinal material extracting solution in the step (2) is prepared by the following steps: the preparation method comprises the steps of drying, crushing and sieving a ginger medicinal material to obtain ginger medicinal material dry powder, adding the ginger medicinal material dry powder into a first solvent according to the material-liquid ratio of 0.05-0.20 g/mL, soaking and extracting for 24-48 hours or ultrasonically extracting for 15-30 minutes, supplementing weight, shaking up and filtering to obtain filtrate, namely the ginger medicinal material extracting solution.
Preferably, the ginger medicinal material preparation extracting solution in the step (2) is prepared by the following steps: adding the ginger medicinal preparation into a first solvent according to the material-to-liquid ratio of 0.01-0.10 g/mL, carrying out ultrasonic extraction for 15-30 minutes, supplementing weight, shaking up, and filtering to obtain a filtrate, namely the ginger medicinal preparation extracting solution.
According to the invention, the N-vanillyl nonanamide reference substance is used as an internal reference substance to establish a one-test-multiple-evaluation method, and the N-vanillyl nonanamide reference substance is similar to a component to be tested in structure, similar in polarity, stable in performance, free of existence in a sample, cheaper and easily available compared with natural capsaicin, has the effects of relieving pain, diminishing inflammation, sterilizing, dispelling wind and dampness and the like, and is more environment-friendly and economical, so that the N-vanillyl nonanamide reference substance is finally preferably used as the internal reference substance.
The gingerol component measured by the invention has similar structure and lower ultraviolet detection response sensitivity, enters MS detection analysis through UPLC separation, has higher sensitivity, and preferably selects aqueous solution of formic acid or acetic acid as a water phase (mobile phase A) and acetonitrile as an organic phase (mobile phase B) to be more suitable for separating the components by comparing the influence of different mobile phase systems on the separation effect.
The separation efficiency of the compounds is greatly influenced by chromatographic columns of different brands, and finally, Eclipse Plus C18 chromatographic columns (100 multiplied by 4.6mm and 3.5 mu m) with higher analysis speed are selected ideally through a large number of experimental screens and an ultra-high performance liquid separation technology, and the flow rate of a mobile phase is screened through a large number of experiments.
The invention aims at the fact that the gingerol component is one of main components contained in the ginger medicinal material, and adopts a one-test-multiple-evaluation technology to establish a method for simultaneously determining 4 representative gingerol component compounds with relatively high content in the ginger medicinal material and preparations thereof. When the relative correction factor is established, two relative correction factor calculation methods which directly calculate the relative correction factor by quantifying the ion peak area and the sample volume and calculate the logarithm of the peak area and the sample volume are compared respectively. The result shows that if the calculation method of the peak area and the sample volume is directly adopted, the relative standard deviation value of the obtained correction factor is larger, and when the logarithm of the peak area and the sample volume is adopted for calculation, the RSD values are all less than 5%, and finally, the calculation of the correction factor is preferably carried out by selecting the logarithm of the peak area and the sample volume.
Compared with the prior art, the invention has the following beneficial effects: according to the structural characteristics of active ingredients and impurity ingredients in ginger medicinal materials, ultrahigh performance liquid chromatography and mass spectrometry analysis conditions are preferably selected through a large number of experiments, a pure substance N-vanillylnonanamide reference substance which does not exist in a sample, is stable in performance, is cheap and easy to obtain is adopted as an internal reference substance to establish a one-test-multiple-evaluation method, relative correction factors of the reference substance and 6-shogaol, 6-gingerol, 8-gingerol and 10-gingerol are measured, and the content of 4 gingerol components is calculated. The method has the advantages of simple operation, high sensitivity, accuracy, high efficiency and low cost, can objectively and accurately evaluate the quality of the ginger medicinal material and the preparation thereof, is used for quality control, can solve the problem that the quality of the ginger medicinal material and the preparation thereof cannot be objectively and reasonably controlled due to the lack of a reference substance, and has important significance for controlling the quality and ensuring the curative effect.
Drawings
FIG. 1 is a TIC and MRM spectra of a mixed control standard solution as described in example 1;
FIG. 2 is a TIC and MRM spectrum of the test solution 2 described in example 1.
Detailed Description
In order to make the technical purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention are further described with reference to specific examples, which are intended to explain the present invention and are not to be construed as limiting the present invention, and those who do not specify a specific technique or condition in the examples follow the techniques or conditions described in the literature in the art or follow the product specification.
The apparatus used in the following examples included: agilent QQQ 6460C-1290UPLC ultra performance liquid chromatography tandem triple quadrupole mass spectrometer (Agilent technologies, inc., usa), AUW220D type electronic analytical balance (Shimadzu, japan), ME 204/one hundred thousandth balance (mettler-toledo, switzerland), QL-901 type vortex mixer (lynbel instruments, inc., haman), pipette: 100. mu.L, 200. mu.L, 1000. mu.L (Eppendorf).
The materials and test samples used in the following examples include: control 6-gingerol (CAS: 23513-14-6, 98.27%) with lot number PCL-G432, purchased from PCL in England; the control 8-gingerol (CAS: 23513-08-8, 93.8%), 10-gingerol (CAS: 23513-15-7, 97.1%), lot numbers 111994-; 6-shogaol (CAS: 555-66-8, 98.20%) as a control, having a batch number of 16122601, purchased from Doppel Biotechnology Ltd; control N-vanillylnonanamide (CAS: 2444-46-4, 98% or more) with batch number wkq16090106, available from Szechwan Vecky Biotech, Inc.; methanol, acetonitrile (LC/MS grade, FisherScientific); formic acid (LC/MS grade, Fisher Scientific); ultrapure water (Wahaha mineral water). Ginger selected from different origins: the method comprises the steps of Zhangliang, Anhui ginger, Shandong ginger and Shandong small ginger, and selecting complete and insect disease-free ginger as a test raw material, wherein the fresh ginger refers to the ginger which does not sweat, and the dried ginger refers to the dried ginger.
Example 1
A method for measuring gingerol content in ginger medicinal material comprises the following steps:
(1) preparation of control solutions
Precisely weighing 5 reference substances including N-vanillylnonanamide, 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol respectively, and dissolving and diluting with methanol to obtain stock solutions of the single reference substances; wherein the concentration of the N-vanillylnonane amide reference stock solution is 509.60 mu g/mL, the concentration of the 6-gingerol reference stock solution is 552.72 mu g/mL, the concentration of the 8-gingerol reference stock solution is 806.68 mu g/mL, the concentration of the 10-gingerol reference stock solution is 592.31 mu g/mL, and the concentration of the 6-gingerol reference stock solution is 509.60 mu g/mL.
Then respectively measuring the single reference substance stock solutions, diluting with methanol, and mixing uniformly to obtain mixed reference substance standard solutions, wherein the concentrations of N-vanillylnonanamide, 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in the mixed reference substance standard solutions are 50.960, 55.272, 80.668, 59.231 and 50.960 mu g/mL respectively; meanwhile, the mass concentration of the preparation is respectively CoLAnd CoHN-Vanillylnonaneamide standard solution of (1), CoL10.20. mu.g/mL, CoH50.96 μ g/mL; and storing the mixed reference substance standard solution and the N-vanillylnonanamide standard solution in a refrigerator at the temperature of-20 ℃ in a dark place.
(2) Preparation of test solution
Cleaning fresh ginger, cutting into slices while the ginger is fresh, drying and crushing at 50 ℃, and sieving by a 50-mesh sieve to obtain dried ginger powder; accurately weighing 10.0g of rhizoma Zingiberis powder, placing into 100mL conical flask with plug, accurately adding 80mL of anhydrous ethanol, sealing, and weighing; leaching at room temperature for 36h, adding anhydrous ethanol for supplementing weight, shaking, and filtering to obtain rhizoma Zingiberis recens extractive solution.
Taking 1mL of ginger medicinal material extract, transferring the ginger medicinal material extract into a 10mL measuring flask, and adding 0.1mL of the ginger medicinal material extract with the concentration C prepared in the step (1)oLThe standard solution of N-vanillylnonanamide is prepared by volume metering and shaking up a solvent (which is prepared by uniformly mixing 0.1 percent formic acid water solution and acetonitrile according to the volume ratio of 80: 20), and filtering through a microporous filter membrane of 0.22 mu m to obtain a test solution 1.
Taking 1mL of ginger medicinal material extract, transferring the ginger medicinal material extract into a 10mL measuring flask, and adding 0.1mL of the ginger medicinal material extract with the concentration C prepared in the step (1)oHThe standard solution of N-vanillylnonanamide is prepared by volume metering and shaking up with a solvent (prepared by evenly mixing 0.1% formic acid water solution and acetonitrile according to the volume ratio of 80: 20), and filtering through a microporous filter membrane of 0.22 mu m to obtain a sample solution 2.
(3) Relative correction factor foxAnd relative retention time RtRIs calculated by
The detection is carried out by using UPLC-MS-DAD (ultra performance liquid chromatography tandem triple quadrupole mass spectrometry detector and ultraviolet detector), and the parameters are set as follows:
1) UPLC Condition
A chromatographic column: eclipse Plus C18 column (100X 4.6mm,3.5 μm)
Flow rate: 0.5mL/min
Column temperature: 35 deg.C
Detection wavelength: 280nm +/-2 nm
Mobile phase: 0.1% aqueous formic acid solution (A) -acetonitrile (B)
Gradient elution:
Figure BDA0001637444630000071
2) MS Condition
An ion source: ESI, detection mode: multiple Reaction Monitoring (MRM), Positive ion mode (Positive) scanning; temperature of the drying gas: 350 ℃; atomizing gas pressure: 275.8 kPa; flow rate of drying gas: 10L/min; capillary voltage: 4.0 kV. The ion pairs of N-vanillyl nonanamide, 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol for qualitative and quantitative analysis, which are detected by adopting an MRM mode, have the following retention time, collision energy and fragmentation voltage:
Figure BDA0001637444630000081
wherein, the ion pairs of the N-vanillyl nonanamide, the 6-gingerol, the 8-gingerol, the 10-gingerol and the 6-shogaol for quantitative analysis are respectively as follows: 294.1([ M + H)]+)→137.0，277.1([M-H2O+H]+)→177.1，305.1→177.1([M-H2O+H]+)，333.2→177.1([M-H2O+H]+) And 277.1 → 137.0.
In the optimization process of 6-gingerol, 8-gingerol and 10-gingerol, [ M + Na ] is easily obtained]+、[M+K]+Not easy to obtain [ M + H]+Ion peak, even if [ M + H ] appears]+Ion peak, but low abundance, not easy to get secondary fragment ion, and [ M + Na]+，[M+K]+Although the abundance of the ions is high, secondary fragments are difficult to obtain, the later qualitative and quantitative requirements cannot be met, and a large number of experimental analyses show that the [ M-H ] is2O+H]+The abundance of ion peak is strong, and the component to be detected can be accurately analyzed, and the corresponding secondary fragment ion can be obtained, so that [ M-H ] is selected2O+H]+The ions are used as the qualitative and quantitative parent ions, and are optimized to screen the qualitative and quantitative daughter ions. The four daughter ions corresponding to each compound are obtained through optimization: n-vanillylnonanamide 137.0, 122.0, 94.1, 141.1; 6-gingerol 177.1, 145.0, 117.1, 137.0; 8-gingerol 177.1, 145.0, 117.1, 115.0; 10-gingerol 117.1, 145.0, 117.1, 137.0; 6-shogaol 137.0, 94.1, 122.0 and 106. Wherein, one with the highest abundance is selected as a quantitative ion, and the other with the highest abundance is selected as a qualitative ion. Thus, the following results are obtained: the ion pairs of N-vanillylnonanamide, 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol for quantitative analysis are respectively as follows: 294.1 → 137.0, 277.1 → 177.1, 305.1 → 177.1, 333.2 → 177.1, 277.1 → 137.0; the ion pairs of N-vanillylnonanamide, 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol used for qualitative analysis are respectively as follows: 294.1 → 122.0, 277.1 → 145.0, 305.1 → 117.1, 333.2 → 145.0, 277.1 → 94.1.
Firstly, taking the mixed reference substance standard solution in the step (1), injecting 0.2, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0 mu L of the mixed reference substance standard solution, and carrying out UPLC-MS-DAD detection to obtain an MRM chromatogram and a TIC chromatogram, wherein the MRM chromatogram and the TIC chromatogram are shown in figure 1; the peak area integration was performed and the obtained linear relationship, correlation coefficient and detection limit were as follows:
Figure BDA0001637444630000091
selecting N-vanillylnonane amide as an internal reference compound, and calculating a relative correction factor and a relative retention time according to a formula.
The correction factor foxThe calculation formula of (2) is as follows:
Figure BDA0001637444630000092
in the formula: a. theoIs the peak area of reference substance N-vanillylnonanamide, CoIs the mass concentration of reference substance N-vanillylnonanamide, AxPeak area of the reference x, CxThe mass concentration of a component to be detected as a reference substance x;
the relative retention time RtRThe calculation formula of (2) is as follows:
Figure BDA0001637444630000093
in the formula: t is tRxThe retention time, t, of the component to be tested, control xRoThe retention time of the reference N-vanillylnonanamide control is shown.
Respectively calculating relative correction factors f of the N-vanillyl nonane amide to 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol by combining peak area dataoxAnd RtRThe results are shown in Table 1.
TABLE 1 relative correction factor f for N-vanillylnonanamide on 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaoloxAnd RtR
Figure BDA0001637444630000094
(4) Determination of active ingredients in test articles
Taking the N-vanillylnonanamide standard solution (the concentration is respectively C) prepared in the step (1)oLAnd CoHThe standard solution of N-vanillylnonanamide) and the sample solutions (sample solution 1 and sample solution 2) prepared in step (2), injecting 5 mu L of sample, and measuring by using UPLC-MS-DAD to obtain a TIC chromatogram and an MRM chromatogram, as shown in FIG. 2; calculating the mass concentrations of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in the test solution according to a formula;
the above formula is:
C'x＝(CxL'+CxH')/2
Figure BDA0001637444630000101
Figure BDA0001637444630000102
Figure BDA0001637444630000103
in the formula: cx' is the mass concentration of the component x to be determined in the test solution, CxL' is a component x to be measured in a test solution at a concentration CoLCalculated mass concentration of N-vanillylnonanamide standard solution, CxH' is a component x to be measured in a test solution at a concentration CoHCalculated mass concentration of N-Vanillylnonanoamide standard solution, Ax' is the peak area of the component x to be measured in the test solution, AoLIs CoLPeak area of N-Vanillylnonanoamide concentration standard solution, AoHIs CoHPeak area of N-vanillylnonanamide standard solution of concentration, foxThe relative correction factor of the N-vanillylnonanamide obtained in the step (3) on the tested components is shown, and N is a concentration influence factor.
After the contents of the components to be measured in the test solution 1 and the test solution 2 are respectively calculated, the contents of the same component to be measured are averaged, and the final measured value of the active component in the test is obtained.
Example 2
A method for measuring the content of gingerol in a ginger medicinal material is carried out according to the steps of example 1, except that the leaching time of 36h is changed into the leaching time of 48h when the test solution in the step (2) is prepared.
Example 3
A method for measuring the content of gingerol in a ginger medicinal material is carried out according to the steps of example 1, except that in the step (2), when the test solution is prepared, the extraction solvent is changed from absolute ethyl alcohol to acetone.
Example 4
A method for measuring the content of gingerol in a ginger medicinal material is carried out according to the steps of example 3, except that the leaching time of 36h is changed into the leaching time of 48h when the test solution in the step (2) is prepared.
Example 5
A method for measuring the content of gingerol in a ginger medicinal material is carried out according to the steps of example 1, except that in the step (2), the extraction solvent is changed from absolute ethyl alcohol to methyl alcohol when the test solution is prepared.
Example 6
A method for measuring the content of gingerol in a ginger medicinal material is carried out according to the steps of example 5, except that the leaching time of 36h is changed into the leaching time of 48h when the test solution in the step (2) is prepared.
The results of the measurements of examples 1 to 6, which were measured and calculated according to the procedures (3) and (4) of example 1, are shown in Table 2.
TABLE 2 comparison of the results of the content determination (mg/g) by the one-test-multiple-evaluation method (QAMS) and the external standard method
Figure BDA0001637444630000111
Note:m: example 1 (ethanol leach 36h),n: example 3 (acetone leach 36h),p: example 5 (methanol leach 36 h).
Example 7
A method for measuring the content of gingerol in a ginger medicinal material preparation is carried out according to the steps of example 1, except that the preparation of the test solution in the step (2) is different.
The test solution is prepared as follows:
grinding the ginger buccal tablets into powder to obtain ginger buccal tablet powder; precisely weighing 0.5g of rhizoma Zingiberis recens tablet powder, placing into a 25mL measuring flask, adding 20mL of 50% methanol aqueous solution into the measuring flask, ultrasonically extracting for 20min, diluting with 50% methanol aqueous solution to scale, shaking, and filtering to obtain rhizoma Zingiberis recens medicinal material preparation extractive solution;
taking 1mL of ginger medicinal preparation extract, transferring to a 5mL measuring flask, and adding 0.1mL of C prepared in the step (1)oLThe standard solution of N-vanillylnonanamide is prepared by diluting the solution to constant volume with a solvent (prepared by uniformly mixing 0.1% formic acid water solution and acetonitrile according to the volume ratio of 80: 20), shaking up, and filtering through a microporous filter membrane of 0.22 mu m to obtain a test solution 1.
Taking 1mL of ginger medicinal preparation extract, transferring to a 5mL measuring flask, and adding 0.1mL of C prepared in the step (1)oHThe standard solution of N-vanillylnonanamide is prepared by diluting the solution to constant volume with a solvent (prepared by uniformly mixing 0.1% formic acid water solution and acetonitrile according to the volume ratio of 80: 20), shaking up, and filtering through a microporous filter membrane of 0.22 mu m to obtain a sample solution 2.
The results of the measurement in example 7, which were measured and calculated according to the steps (3) and (4) in example 1, are shown in Table 3.
TABLE 3 comparison of the results of the content determination (mg/g) by the one-test-multiple-evaluation method (QAMS) and the external standard method
Figure BDA0001637444630000121
Example 8
A method for measuring the content of gingerol in a ginger medicinal material preparation is carried out according to the steps of example 1, except that the preparation of the test solution in the step (2) is different.
The test solution is prepared as follows:
weighing 2.0g of ginger granules precisely, placing the ginger granules into a 50mL centrifuge tube, adding 30mL of 50% methanol solution into a conical flask, weighing the mass, carrying out ultrasonic extraction for 20min, cooling to room temperature, supplementing the weight with methanol, centrifuging for 10min at 6000r/min, taking supernatant, and filtering to obtain a ginger medicinal material preparation extracting solution;
taking 1mL of ginger medicinal preparation extract, transferring to a 5mL measuring flask, and adding 0.1mL of C prepared in the step (1)oLThe standard solution of N-vanillylnonanamide is prepared by diluting the solution to constant volume with a solvent (prepared by uniformly mixing 0.1% formic acid water solution and acetonitrile according to the volume ratio of 80: 20), shaking up, and filtering through a microporous filter membrane of 0.22 mu m to obtain a test solution 1.
Taking 1mL of ginger medicinal preparation extract, transferring to a 5mL measuring flask, and adding 0.1mL of C prepared in the step (1)oHThe standard solution of N-vanillylnonanamide is prepared by diluting the solution to constant volume with a solvent (prepared by uniformly mixing 0.1% formic acid water solution and acetonitrile according to the volume ratio of 80: 20), shaking up, and filtering through a microporous filter membrane of 0.22 mu m to obtain a sample solution 2.
The results of the measurement in example 8, which were measured and calculated according to the steps (3) and (4) in example 1, are shown in Table 3.
TABLE 4 comparison of the results of content determination (mg/g) by the one-test-multiple-evaluation method (QAMS) and the external standard method
Figure BDA0001637444630000122
The experimental results show that the method for measuring the content of gingerol in the ginger medicinal material and the preparation thereof provided by the invention is simple to operate, high in sensitivity, accurate, efficient and low in cost, can objectively and accurately evaluate the quality of the ginger medicinal material and the preparation thereof, and is used for quality control, and experimental comparison results show that the method is equivalent to the detection result of an external standard method in accuracy, but the method provided by the invention adopts a one-test-multiple evaluation method, is more efficient and faster, adopts the N-vanillylnonanamide which does not exist in a sample as an internal reference substance, is similar in performance, stable and easy to obtain, and is more economic.
The invention optimizes the optimum analysis conditions of mobile phase gradient elution, flow velocity, chromatographic column, mass spectrum parameters and the like and volatile oil extraction conditions through a large number of experiments, adopts UPLC separation, MS ion pair qualitative, DAD qualitative and more accurate qualitative, MS quantitative detection, establishes relative correction factors of N-vanillyl nonanamide with 6-gingerol, 8-gingerol, 10-gingerol and 6-gingerol by taking N-vanillyl nonanamide as an internal reference through a one-test-multiple-evaluation technology, and calculates the content of 4 gingerol components, thereby solving the problem that the quality of ginger medicinal materials and preparations thereof cannot be objectively and reasonably controlled due to the lack of reference substances, and obtaining better technical effects.
The above description is only a preferred embodiment of the present invention, and it should be understood that any modification, equivalent replacement and modification made by those skilled in the art without departing from the principle of the present invention shall be included in the protection scope of the present invention.

Claims (8)

1. A method for measuring gingerol content in ginger medicinal material and preparations thereof is characterized by comprising the following steps:
(1) preparation of control solutions
Precisely weighing 5 reference substances, namely N-vanillylnonanamide, 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol, respectively, dissolving and diluting with methanol to obtain single reference substance stock solutions, measuring the single reference substance stock solutions, diluting with methanol and uniformly mixing to obtain mixed reference substance standard solutions; meanwhile, the mass concentration of the preparation is respectively CoLAnd CoHN-Vanillylnonaneamide standard solution of (1), CoLLess than CoH(ii) a Freezing and storing the mixed reference substance standard solution and the N-vanillylnonanamide standard solution in a dark place;
(2) preparation of test solution
Weighing a ginger medicinal material or a ginger medicinal material preparation, adding the ginger medicinal material and the ginger medicinal material preparation into a first solvent respectively for extraction to obtain a ginger medicinal material extracting solution and a ginger medicinal material preparation extracting solution, and taking the ginger medicinal material extracting solution or the ginger medicinal material preparation extracting solution as a sample;
directly sampling a sample, adding the N-vanillyl nonane amide reference substance stock solution or the N-vanillyl nonane amide standard solution prepared in the step (1), mixing, and fixing the volume by using a second solvent to obtain a sample solution;
or diluting a sample with 50-100% methanol to obtain a sample mother solution, then sampling the sample mother solution, adding the N-vanillyl nonanamide reference substance stock solution or the N-vanillyl nonanamide standard solution prepared in the step (1), mixing, and fixing the volume with a second solvent to obtain a sample solution; the content of the N-vanillylnonanamide in the test solution is 0.05-1.00 mu g/mL;
(3) relative correction factor foxAnd relative retention time RtRIs calculated by
Measuring by using UPLC-MS-DAD, sampling the mixed reference standard solution obtained in the step (1) of the series concentration, obtaining a TIC chromatogram and an MRM chromatogram, carrying out peak area integration, selecting N-vanillyl nonanamide as an internal reference compound, and respectively calculating the relative correction factors f of the N-vanillyl nonanamide to 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in the mixed reference standard solutionoxAnd relative retention time RtR(ii) a The relative correction factor foxThe calculation formula of (2) is as follows:
Figure 481557DEST_PATH_IMAGE001
in the formula: a. theoIs the peak area of reference substance N-vanillylnonanamide, CoIs the mass concentration of reference substance N-vanillylnonanamide, AxPeak area of the reference x, CxThe mass concentration of a component to be detected as a reference substance x; the relative retention time RtRThe calculation formula of (2) is as follows:
Figure 611187DEST_PATH_IMAGE002
in the formula: t is tRxThe retention time, t, of the component to be tested, control xRoThe retention time of the reference substance N-vanillylnonanamide is shown;
(4) determination of active ingredients in test articles
Sampling the N-vanillyl nonanamide standard solution prepared in the step (1) and the test sample solution prepared in the step (2), determining by using UPLC-MS-DAD to obtain a TIC chromatogram and an MRM chromatogram, and calculating the mass concentrations of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in the test sample solution according to a formula;
the above formula is:
Figure 946983DEST_PATH_IMAGE003
in the formula: cx' is the mass concentration of the component x to be determined in the test solution, CxL' is a component x to be measured in a test solution at a concentration CoLCalculated mass concentration of N-vanillylnonanamide standard solution, CxH' is a component x to be measured in a test solution at a concentration CoHCalculated mass concentration of N-Vanillylnonanoamide standard solution, Ax' is the peak area of the component x to be measured in the test solution, AoLIs CoLPeak area of N-Vanillylnonanoamide concentration standard solution, AoHIs CoHPeak area of N-vanillylnonanamide standard solution of concentration, foxThe relative correction factor of the N-vanillylnonanamide obtained in the step (3) to the tested components, wherein N is a concentration influence factor;
the conditions for UPLC determination in the step (3) and the step (4) are as follows: the chromatographic column is a reversed phase C18 chromatographic column, the mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is aqueous solution of formic acid with volume concentration of 0.02-0.2% or aqueous solution of acetic acid with volume concentration of 0.02-0.2%, acetonitrile is used as the mobile phase B, gradient elution is carried out, the flow rate is 0.4-0.6 mL/min, and the detection wavelength is 280nm +/-2 nm;
the procedure for the gradient elution was:
Figure 707128DEST_PATH_IMAGE004
。
2. the method for measuring the content of gingerol in the ginger medicinal material, the ginger extract and the ginger preparation according to claim 1, is characterized in that: and (3) uniformly mixing the mobile phase A and the mobile phase B according to the initial proportion of gradient elution to obtain the second solvent in the step (2).
3. The method for measuring the content of gingerol in the ginger medicinal material and the preparation thereof according to claim 1, wherein the conditions of MS measurement in the steps (3) and (4) are as follows: the ion source is ESI, the detection mode is MRM multi-reaction monitoring, the scanning mode is a positive ion mode, the pressure of atomizing gas is 275.8-379.2 kPa, the temperature of drying gas is 350 ℃, the flow rate of drying gas is 10-12L/min, and the voltage of a capillary is 4.0 kV.
4. The method for determining gingerol content in ginger and its preparations according to claim 3, wherein the MS determination conditions further include: ion pairs for quantitative analysis: n-vanillylnonanamide 294.1 → 137.0, 6-gingerol 277.1 → 177.1, 8-gingerol 305.1 → 177.1, 10-gingerol 333.2 → 177.1, 6-shogaol 277.1 → 137.0.
5. The method for determining gingerol content in ginger and its preparations according to claim 3, wherein the MS determination conditions further include: ion pairs for qualitative analysis: n-vanillylnonanamide 294.1 → 122.0, 6-gingerol 277.1 → 145.0, 8-gingerol 305.1 → 117.1, 10-gingerol 333.2 → 145.0, 6-shogaol 277.1 → 94.1.
6. The method for measuring the content of gingerol in the ginger medicinal material and the preparation thereof according to claim 1, is characterized in that: in the step (2), the first solvent is one of absolute ethyl alcohol, acetone, methanol and water.
7. The method for measuring the content of gingerol in the ginger medicinal material and the preparation thereof according to claim 1, wherein the ginger medicinal material extracting solution in the step (2) is prepared by the following steps: the preparation method comprises the steps of drying, crushing and sieving a ginger medicinal material to obtain ginger medicinal material dry powder, adding the ginger medicinal material dry powder into a first solvent according to the material-liquid ratio of 0.05-0.20 g/mL, soaking and extracting for 24-48 hours or ultrasonically extracting for 15-30 minutes, supplementing weight, shaking up and filtering to obtain filtrate, namely the ginger medicinal material extracting solution.
8. The method for measuring the content of gingerol in the ginger medicinal material and the preparation thereof according to claim 1, wherein the ginger medicinal material preparation extracting solution in the step (2) is prepared by the following steps: adding the ginger medicinal preparation into a first solvent according to the material-to-liquid ratio of 0.01-0.10 g/mL, carrying out ultrasonic extraction for 15-30 minutes, supplementing weight, shaking up, and filtering to obtain a filtrate, namely the ginger medicinal preparation extracting solution.
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