CN103604890A - Schisandra fruit organic acid analysis method - Google Patents
Schisandra fruit organic acid analysis method Download PDFInfo
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Abstract
The invention discloses a schisandra fruit organic acid analysis method. The invention provides a method for analyzing organic acid in a schisandra fruit processed product by using GC-MS (gas chromatography-mass spectrometry), and a method for measuring the content of 4-oxo-valeric acid in a schisandra fruit processed product. The analysis method comprises the following steps: taking schisandra fruit powder, adding sulfuric acid methanol, NaCl, an aether-chloroform mixed solution and anhydrous sodium sulfate, and drying to obtain a sample; and carrying out GC-MS analysis. The analysis method has the advantages of high specificity and favorable reproducibility, and provides references for quality control on organic acid before and after schisandra fruit processing.
Description
Technical field
The present invention relates to the field of quality control of the prepared slices of Chinese crude drugs, be specifically related to fruit of Chinese magnoliavine medicine materical crude slice organic acid analytical approach and the method for quality control of representative substances LA wherein.
Background technology
The fruit of Chinese magnoliavine is the fruit of the drying and ripening of Magnoliaceae fruit of Chinese magnoliavine Schisandra chinese (Turcz.) Baill., has convergence astringent or styptic treatment for spontaneous sweating, nourishing generate fluid, the effect of kidney calming.Clinically for the void of coughing for a long time, breathe heavily, emission, enuresis frequent micturition, endless diarrhea, spontaneous perspiration, night sweat, Tianjin wound is thirsty, the feeble pulse of losing heart, interior heat is quenched one's thirst, palpitation and insomnia.The process of preparing Chinese medicine kind of 2010 editions fruit ofs Chinese magnoliavine of recording of < < Chinese Pharmacopoeia > > is that the fruit of Chinese magnoliavine and vinegar steam the fruit of Chinese magnoliavine, in the clinical practice of the fruit of Chinese magnoliavine, also take these two kinds as main, vinegar is sour and astringent effect enhancing after steaming, and puckery smart anti-diarrhea effect strengthens.In the fruit of Chinese magnoliavine, principal ingredient comprises lignanoid, volatile oil, organic acid etc.Wherein, the composition of lignanoid is mainly cyclohexyl biphenyl octene type lignanoid, modern study thinks that it is topmost bioactive ingredients in the fruit of Chinese magnoliavine more, have and the pharmacological action such as protect the liver, anti-oxidant, take schizandrin, wuweizi alcohol B, schizandrin A, deoxyschizandrin, schisandrin C etc. is representative, wave with oil mainly with australene, amphene, nopinene, laurene, α-terpinene, citrene, γ-terpinene etc. are main, and organic acid is main mainly with small molecular organic acid, as citric acid, malic acid, tartrate, succinic acid etc.
Pharmacopeia and each research report mainly concentrate on the research to its contained lignans at present, the lignanoid's content that has more document to concoct front and back to the fruit of Chinese magnoliavine is analyzed, its presentation of results fruit of Chinese magnoliavine Jing Cuzhenghou lignanoid content has increased slightly, but basic there was no significant difference, in existing version pharmacopeia about the representative composition schizandrin of quality control Jun Zhiyi lignanoid of fruit of Chinese magnoliavine medicine materical crude slice and steamed FRUCTUS SCHISANDRAE with vinegar medicine materical crude slice as its index composition, and limit is identical.But drug action and the clinical application of the fruit of Chinese magnoliavine before and after vinegar steams has larger difference, and fruit of Chinese magnoliavine vinegar steam after major embodiment be that anti-diarrhea effect strengthens, there is no the bibliographical information that it strengthens the Related Component of antidiarrheal.Therefore fruit of Chinese magnoliavine processed product is carried out to comprehensive quality assessment, particularly select with drug action Related Component and determine its limit, could more effectively guarantee the quality of fruit of Chinese magnoliavine processed product, to guarantee that it brings into play maximum effect clinically.
As the representative of convergence astringent drug, the Determination of Organic Acids in the fruit of Chinese magnoliavine also can not be ignored.The drug action of the fruit of Chinese magnoliavine should be multicomponent combined action, is not only embodied in its main lignan component, therefore to organic acid chemical composition, pharmacological action in the fruit of Chinese magnoliavine, also should further investigate the difference before and after particularly concocting on organic acid.What on document, to fruit of Chinese magnoliavine organic acid, analysis was mainly taked is its contained total organic acids content of potentiometric determination, and less to its concrete Determination of Organic Acids report, the variation that particularly vinegar steams rear Determination of Organic Acids is without bibliographical information.
The present invention is directed to the blank of existing research, adopt new means to carry out quality assessment to the organic acid in the fruit of Chinese magnoliavine, particularly the raw fruit of Chinese magnoliavine and steamed FRUCTUS SCHISANDRAE with vinegar organic acid have been taked the method for direct esterification, and adopt the method for gas chromatography mass spectrometry to carry out comprehensive analysis, the content of finding the rear LA of fruit of Chinese magnoliavine vinegar steaming in research process obviously increases, by pharmacological evaluation, prove that LA has the effect of antidiarrheal, measured the content of LA wherein and stipulated limit, solved a difficult problem for fruit of Chinese magnoliavine processed product organic acid quality control, fully demonstrate the index relevant to drug effect and controlled the requirement of prepared slice quality, organic acid quality testing after raising can be controlled the organic acid quality in fruit of Chinese magnoliavine processed product better, this method has stronger specificity and good reappearance.
Summary of the invention
In order to address the above problem, to the object of the present invention is to provide a kind of method of fruit of Chinese magnoliavine organic acid analysis detection, and set up the content assaying method of LA in fruit of Chinese magnoliavine processed product.In order to reach this object, adopt following technical scheme:
A fruit of Chinese magnoliavine processed product organic acid analytical approach, comprises the following steps:
(1) sample preparation methods: get the raw fruit of Chinese magnoliavine (60 order), each about 0.2g of steamed FRUCTUS SCHISANDRAE with vinegar (60 order), accurately weighed, put in tool plug test tube, precision adds sulfuric acid methyl alcohol 1.0mL 35 ℃ of vibration 24h(150r/min in constant temperature oscillator of 10%) after, precision adds saturated NaCl solution 2mL and E-C (2:1) mixed liquor 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
(2) GC-M S analysis condition: mass spectrum condition: EI ion gun; Electron energy 70eV; 230 ℃ of ion source temperatures; 150 ℃ of level Four bar temperature; Detect voltage 1235V; Solvent delay 3.2min.Chromatographic condition: HP-5 chromatographic column (30m * 250 μ m, 0.5 μ m); Helium (99.999%) is carrier gas, flow velocity 1.0mL/min; Before post, press 0.171MPa; 250 ℃ of injector temperatures; Split sampling (split ratio 15:1); Temperature programme: 40 ℃ of initial temperatures, rise to 260 ℃ (constant temperature 10min) with the speed of 5 ℃/min.
Adopt said method to analyze fruit of Chinese magnoliavine processed product organic acid, result shows: after fruit of Chinese magnoliavine vinegar steams, LA its relative content in raw fruit of Chinese magnoliavine medicine materical crude slice is 0.692%, and relative content reaches 1.653% in steamed FRUCTUS SCHISANDRAE with vinegar medicine materical crude slice, in steamed FRUCTUS SCHISANDRAE with vinegar, LA relative content is 2.38 times in the raw fruit of Chinese magnoliavine.
The present invention also discloses a kind of content assaying method that utilizes the LA of above-mentioned organic acid analysis method, and the method comprises the following steps:
(1) preparation of reference substance solution: the accurate LA reference substance of drawing is put in 5mL measuring bottle in right amount respectively, respectively adds 10% sulfuric acid dissolve with methanol solution and is settled to scale.
(2) the preparation precision of inner mark solution takes gaultherolin in right amount to 100mL measuring bottle, with E-C mixed liquor (2:1), dissolves and is diluted to scale.
(3) fruit of Chinese magnoliavine medicine materical crude slice powder (60 order) 0.2g is got in the preparation of need testing solution, accurately weighed, put in tool plug test tube, precision adds 10% sulfuric acid methyl alcohol 1.0mL solution, be placed in 35 ℃ of vibration 24h(150r/min of constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C mixed liquor (2:1) 2mL, DL 3min, centrifugal 5min (3000r/min), the accurate supernatant 1mL that draws, adds gaultherolin inner mark solution 1mL, adds appropriate anhydrous sodium sulfate, dry, obtain.
(4) GC-MS analysis condition mass spectrum condition: EI ion gun; Electron energy 70eV; 230 ℃ of ion source temperatures; 150 ℃ of level Four bar temperature; Detect voltage 1235V; Solvent delay 3.2min.Chromatographic condition: HP-5 chromatographic column (μ m * 0.5,30m * 250 μ m); Helium (99.999%) is carrier gas, flow velocity 1.0mL/min; Before post, press 0.171MPa; 250 ℃ of injector temperatures; Split sampling (split ratio 15:1); Temperature programme: 40 ℃ of initial temperatures, rise to 140 ℃ with the speed of 5 ℃/min.
(5) assay: accurate reference substance and the need testing solution 1 μ l of drawing, inject gas chromatograph, measures (n=3), obtains.
The invention also discloses a kind of fruit of Chinese magnoliavine method of quality control, is to measure LA content in the fruit of Chinese magnoliavine with said method, and according to LA content meter in fruit of Chinese magnoliavine dry product, result is no less than setting value.The setting value of the raw fruit of Chinese magnoliavine and steamed FRUCTUS SCHISANDRAE with vinegar is respectively 0.28%, 0.60%.The present invention has carried out comprehensive analysis and has proposed method of quality control fruit of Chinese magnoliavine organic acid, the method provides the content of LA composition in Drugs by GC, and method of quality control of the present invention has stronger specificity and good reappearance.
Accompanying drawing explanation
Fig. 1 is fruit of Chinese magnoliavine processed product organic acid methyl ester GC-MS total ions chromatogram.
Fig. 2 is fruit of Chinese magnoliavine processed product organic acid position esterification GC-MS total ions chromatogram.
Fig. 3 is LA methyl esters mass spectrogram.
Fig. 4 is LA content measuring standard curve map.
Fig. 5 is fruit of Chinese magnoliavine processed product LA assay GC-MS total ion current figure.
Embodiment
In conjunction with embodiment, the present invention is further described as follows:
1.1 instruments and reagent
1.1.1 instrument
U.S. Agilent GC6890-MS type gas chromatograph-mass spectrometer (GCMS) (being furnished with NIST2005 standard spectrum storehouse), LXJ-2B Multi-pipe centrifugal machine (Anting Scientific Instrument Factory, Shanghai), METTLER AE240 electronic analytical balance (Mei Teletuo benefit Instrument Ltd.), the desk-top constant temperature oscillator of THZ-D (Taicang experimental facilities factory).
1.1.2 reagent
It is pure that methyl alcohol, the concentrated sulphuric acid, chloroform, ether, anhydrous sodium sulfate, sodium chloride are analysis.
The preparation of 1.2 samples
1.2.1 the preparation of fruit of Chinese magnoliavine medicine materical crude slice esterification sample
Get the raw fruit of Chinese magnoliavine (60 order), each about 0.2g of steamed FRUCTUS SCHISANDRAE with vinegar (60 order), accurately weighed, put in tool plug test tube, precision adds sulfuric acid methyl alcohol 1.0mL 35 ℃ of vibration 24h(150r/min in constant temperature oscillator of 10%) after, precision adds saturated NaCl solution 2mL and E-C (2:1) mixed liquor 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.2.2 the preparation of medicine materical crude slice sample blank
Get the raw fruit of Chinese magnoliavine (60 order), each about 0.2g of steamed FRUCTUS SCHISANDRAE with vinegar (60 order), accurately weighed, put in tool plug test tube, precision adds methyl alcohol 1.0mL 35 ℃ of vibration 24h(150r/min in constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C (2:1) mixed liquor 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.2.3 the preparation of vinegar esterification sample
Get 10mL vinegar and by vinegar, steam the processing procedure of the fruit of Chinese magnoliavine, normal pressure steams after 5h, add water and be settled to 25mL, the accurate 0.1mL solution (amount that is equivalent to the auxiliary material vinegar that adds in 0.2g medicinal material) of drawing, put in tool plug test tube, precision adds sulfuric acid methyl alcohol 1.0mL 35 ℃ of vibration 24h(150r/min in constant temperature oscillator of 10%) after, precision adds saturated NaCl solution 2mL and E-C (2:1) mixed liquor 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.2.4 the preparation of vinegar sample blank
Get 10mL vinegar and by vinegar, steam the processing procedure of the fruit of Chinese magnoliavine, normal pressure steams after 5h, add water and be settled to 25mL, the accurate 0.1mL solution (amount that is equivalent to the auxiliary material vinegar that adds in 0.2g medicinal material) of drawing, put in tool plug test tube, precision adds methyl alcohol 1.0mL 35 ℃ of vibration 24h(150r/min in constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C (2:1) mixed liquor 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.2.5 the preparation of reagent esterification blank
The sulfuric acid methyl alcohol 1.0mL solution of accurate absorption 10% is put in tool plug test tube, 35 ℃ of vibration 24h(150r/min in constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C mixed liquor (2:1) 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.2.6 the preparation of reagent blank
The accurate methyl alcohol 1.0mL that draws puts in tool plug test tube, 35 ℃ of vibration 24h(150r/min in constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C mixed liquor (2:1) 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.3GC-M S analysis condition
Mass spectrum condition: EI ion gun; Electron energy 70eV; 230 ℃ of ion source temperatures; 150 ℃ of level Four bar temperature; Detect voltage 1235V; Solvent delay 3.2min.
Chromatographic condition: HP-5 chromatographic column (30m * 250 μ m, 0.5 μ m); Helium (99.999%) is carrier gas, flow velocity 1.0mL/min; Before post, press 0.171MPa; 250 ℃ of injector temperatures; Split sampling (split ratio 15:1); Temperature programme: 40 ℃ of initial temperatures, rise to 260 ℃ (constant temperature 10min) with the speed of 5 ℃/min.
Each need testing solution 1.0 μ L are carried out to GC-MS analysis by 1.3 lower conditions.
1.4 result
In experimentation, esterification reaction of organic acid condition has been carried out preferably: compared respectively methyl esterification reagent 10% sulfuric acid methanol solution and benzene-10% sulfuric acid methanol solution (1:1), sulfuric acid concentration 8% sulfuric acid-methyl alcohol, 10% sulfuric acid-methyl alcohol, 12% sulfuric acid-methyl alcohol, consumption 1mL, the 2mL of methyl esterification reagent, 3mL, 12 hours esterification time, 24 hours, 36 hours, according to GC analysis result, number and the abundance at peak of take is index, determines that 10% sulfuric acid-methyl alcohol 1mL vibration esterification in 24 hours is substantially complete; Extraction solvent benzene and chloroform-ether are compared, find take that benzene is as extracting the chromatogram of solvent, solvent appearance time is late, for fear of the interference to Determination of Organic Acids, selects chloroform-ether (2:1) as extraction solvent; For promoting esterification liquid separatory, added therein saturated nacl aqueous solution, and to its usage ratio carried out being 1,1.5mL, 2mL investigate, and determines that 2mL saturated sodium-chloride meets acidity requirement.
Fruit of Chinese magnoliavine processed product organic acid methyl ester compound and blank sample are carried out to separation, evaluation with gas chromatograph-mass spectrometer, through retrieving with the contrast of NIST2005 standard spectrum storehouse, result detects 69 from life, steamed FRUCTUS SCHISANDRAE with vinegar medicine materical crude slice, 70 kind of composition, accounts for respectively 93.360%, 92.226% of its total amount.Concrete outcome is in Table 1 and Fig. 1.
The result detecting from table 1 can find out, the organic acid of the raw fruit of Chinese magnoliavine and steamed FRUCTUS SCHISANDRAE with vinegar forms and has certain similarity in chemical composition.Meanwhile, in the relative scale that both are grouped at kind and the one-tenth of chemical composition, all there is certain difference.
Some composition cannot detect after fruit of Chinese magnoliavine vinegar steams, and in raw fruit of Chinese magnoliavine medicine materical crude slice, has, and does not detect in corresponding vinegar product, as the dimethyl malenate of 9.478min (percentage composition in the raw fruit of Chinese magnoliavine is 0.033%).
But simultaneously, after vinegar steams, have being detected of some new compounds, the characteristic peak if the 2 hydroxy propanoic acid methyl esters of 4.522min is vinegar, all detects this thing in steamed FRUCTUS SCHISANDRAE with vinegar and auxiliary material vinegar, and in corresponding raw product all without this thing.The Alpha-hydroxy methyl phenylpropionate of 22.807min (percentage composition in steamed FRUCTUS SCHISANDRAE with vinegar is 0.041%), the unknown material of 32.89min (percentage composition in steamed FRUCTUS SCHISANDRAE with vinegar is 0.029%), the unknown material of 39.148min (percentage composition in steamed FRUCTUS SCHISANDRAE with vinegar is 0.076%), above material does not all detect in the raw fruit of Chinese magnoliavine, therefore illustrates that this material may be that vinegar steaming heating process produces.
But more is that composition has produced variation difference on content after vinegar steams.After vinegar steams, some increased contents, as 2 of 10.184min, 2-dimethoxy methyl propionate (crude drug 0.016%, vinegar medicine 0.084%); 11.46min4-oxopentanoic acid methyl esters (crude drug 0.692%, vinegar medicine 1.653%); The unknown material of 14.409min (the little not integration of crude drug peak amount, vinegar medicine 0.021%); The unknown material of 16.631min (crude drug 1.67%, vinegar medicine 3.284%); The unknown material of 19.646min (crude drug 0.213, vinegar medicine 0.323%); The unknown material of 21.536min (crude drug 0.006%, vinegar medicine 0.019%); The unknown material of 21.598min (crude drug 0.016%, vinegar medicine 0.024%); The unknown material of 29.71min (crude drug 0.031%, vinegar medicine 0.043%); The 12-methyl tridecylic acid methyl esters (crude drug 0.068%, vinegar medicine 0.105%) of 31.163min; 33.063 unknown material (crude drug 0.199%, vinegar medicine 0.516%); The pentadecanoic acid methyl esters of 33.316min (crude drug 0.067%, vinegar medicine 0.11%); 33.473 unknown material (crude drug 0.013%, vinegar medicine 0.031%); The methyl margarate of 37.371min (crude drug 0.021%, vinegar medicine 0.038%); The unknown material of 39.711min (crude drug 0.366%, vinegar medicine 0.533%).Also after having vinegar to steam, content reduces, and be mainly compound and the unknown compound of some non-acid, and the relative content of these compounds is all less than 0.2%, as 1-propylene-1 of the unknown material of 22.431min, 24.372min, 1 of 2,3-tricarboxylic acids trimethyl, 25.732min, 2,3,4,4a, 5,6,8a-octahydro-4a, 8-dimethyl-2-(1-the first and second bases)-naphthalene; 25.87min methyl n-undecyl ketone; 1-methyl-4-(1 of 26.461min, 2,2-trimethyl cyclopentyl) benzene; 26.812min 1,6-dimethyl-4-(1-Methylethyl)-1,2,3,1,2 of α-calacorene of 4-naphthane, 27.326min, 29.187min, 4a, 5,8,8a-, six hydrogen-4,7-dimethyl-1-(1-the first and second bases)-naphthalene; 29.483min aromadendrene; The 1A of 29.925min, 2,3,4,4A, 5,6,7B-octahydro-1, Isosorbide-5-Nitrae, 7-tetramethyl-1H-cyclopropylene is also difficult to understand; 2,6,6 of 30.096min, 9-tetramethyl-tri-encircle [5.4.0.0 (2,8)] 11-9-alkene; 30.251min unknown material; 6-dimethyl-4-(1-Methylethyl of 30.427min) naphthalene; 30.669 2,4a, 5,6,9a-, six hydrogen-3,5,5,9-tetramethyl (1H) benzo ring heptene; 1,5,5 of 30.872min, 9-tetramethyl-1,8-diene-spiral shell [55] undecane; 32.171min unknown material; 32.514min unknown material; 33.563 unknown material; 33.96 unknown material; 36.907min unknown material; 39.064 unknown material.
GC-MS total ion current figure from the esterification of fruit of Chinese magnoliavine sample, after fruit of Chinese magnoliavine vinegar steams, LA its relative content in raw fruit of Chinese magnoliavine medicine materical crude slice is 0.692%, and relative content reaches 1.653% in steamed FRUCTUS SCHISANDRAE with vinegar medicine materical crude slice, steamed FRUCTUS SCHISANDRAE with vinegar organic acid relative content is that raw fruit of Chinese magnoliavine organic acid relative content has 2.38 times, points out it may be corresponding with the drug action of the fruit of Chinese magnoliavine after vinegar steams.
The different processed product organic acid of table 1 fruit of Chinese magnoliavine GC-MS analysis result
Note *: after the esterification of auxiliary material vinegar, have this peak
&: have this peak in reagent esterification or reagent blank
#: have this peak, but measure little not integration
△: all have this peak in raw fruit of Chinese magnoliavine blank and steamed FRUCTUS SCHISANDRAE with vinegar blank sample
The analysis comparison at embodiment 2, fruit of Chinese magnoliavine processed product organic acid position
1.1 instruments and reagent
1.1.1 instrument
U.S. Agilent GC6890-MS type gas chromatograph-mass spectrometer (GCMS) (being furnished with NIST2005 standard spectrum storehouse), LXJ-2B Multi-pipe centrifugal machine (Anting Scientific Instrument Factory, Shanghai), METTLER AE240 electronic analytical balance (Mei Teletuo benefit Instrument Ltd.), the desk-top constant temperature oscillator of THZ-D (Taicang experimental facilities factory).
1.1.2 reagent
It is pure that methyl alcohol, the concentrated sulphuric acid, chloroform, ether, anhydrous sodium sulfate, sodium chloride are analysis.
The preparation of 1.2 samples
1.2.1 the preparation of organic acid position esterification sample
Get raw fruit of Chinese magnoliavine organic acid position, each about 50mg of steamed FRUCTUS SCHISANDRAE with vinegar organic acid position, accurately weighed, put in tool plug test tube, precision adds sulfuric acid methyl alcohol 1.0mL 35 ℃ of vibration 24h(150r/min in constant temperature oscillator of 10%) after, precision adds saturated NaCl solution 2mL and E-C (2:1) mixed liquor 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.2.2 the preparation of organic acid position sample blank
Get raw fruit of Chinese magnoliavine organic acid position, each about 50mg of steamed FRUCTUS SCHISANDRAE with vinegar organic acid position, accurately weighed, put in tool plug test tube, precision adds methyl alcohol 1.0mL 35 ℃ of vibration 24h(150r/min in constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C (2:1) mixed liquor 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.2.3 the preparation of reagent esterification blank
The sulfuric acid methyl alcohol 1.0mL solution of accurate absorption 10% is put in tool plug test tube, 35 ℃ of vibration 24h(150r/min in constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C mixed liquor (2:1) 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.2.4 the preparation of reagent blank
The accurate methyl alcohol 1.0mL that draws puts in tool plug test tube, 35 ℃ of vibration 24h(150r/min in constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C mixed liquor (2:1) 2mL, DL 3min, centrifugal 5min (3000r/min), gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain.
1.3GC-M S analysis condition
Mass spectrum condition: EI ion gun; Electron energy 70eV; 230 ℃ of ion source temperatures; 150 ℃ of level Four bar temperature; Detect voltage 1235V; Solvent delay 3.2min.
Chromatographic condition: HP-5 chromatographic column (30m * 250 μ m, 0.5 μ m); Helium (99.999%) is carrier gas, flow velocity 1.0mL/min; Before post, press 0.171MPa; 250 ℃ of injector temperatures; Split sampling (split ratio 15:1); Temperature programme: 40 ℃ of initial temperatures, rise to 260 ℃ (constant temperature 10min) with the speed of 5 ℃/min.
Each need testing solution 1.0 μ L are carried out to GC-MS analysis by 1.3 lower conditions.
1.4 results and discussion
Fruit of Chinese magnoliavine processed product organic acid position methyl esters compound and blank sample are carried out to separation, evaluation with gas chromatograph-mass spectrometer, through retrieving with the contrast of NIST2005 standard spectrum storehouse, result detects 68 from the raw fruit of Chinese magnoliavine, steamed FRUCTUS SCHISANDRAE with vinegar organic acid position, 76 kind of composition, accounts for respectively 91.347%, 84.110% of its total amount.Concrete result for retrieval is in Table 1, and the GC-MS total ion current figure of each sample of the fruit of Chinese magnoliavine is shown in respectively Fig. 2.
The result detecting from table 1 can find out, the raw product of the fruit of Chinese magnoliavine have certain similarity with organic acid position and medicine materical crude slice that vinegar steams product at chemical composition composition.Meanwhile, in the relative scale that both are grouped at kind and the one-tenth of chemical composition, all there is certain difference.
The assay of LA in embodiment 3, the different processed products of the fruit of Chinese magnoliavine
1 instrument and reagent
1.1 instrument U.S. Agilent GC6890-MS type gas chromatograph-mass spectrometer (GCMS)s (being furnished with NIST2005 standard spectrum storehouse), LXJ-2B Multi-pipe centrifugal machine (Anting Scientific Instrument Factory, Shanghai), METTLER AE240 electronic analytical balance (Mei Teletuo benefit Instrument Ltd.), the desk-top constant temperature oscillator of THZ-D (Taicang experimental facilities factory).
1.2 reagent
It is pure that methyl alcohol, the concentrated sulphuric acid, chloroform, ether, anhydrous sodium sulfate, sodium chloride, salicylic acid are analysis.
1.3 reference substance
LA is purchased from Tokyo HuaCheng Industry Co., Ltd.
2. method and result
The confirmation of 2.14-oxopentanoic acid is carried out esterification by LA reference substance with reference to relevant fruit of Chinese magnoliavine esterification method and is injected GC-MS and analyze, result shows that standard items go out peak in 11.46min4-oxopentanoic acid methyl esters with the time, consistent with the appearance time of the LA methyl esters of confirming after fruit of Chinese magnoliavine esterification, and confirm as same compound through mass spectrum.Its mass spectrogram is shown in Fig. 3.
The preparation of the 2.2 reference substance solution respectively accurate LA reference substance of drawing is put in 5mL measuring bottle in right amount, respectively adds 10% sulfuric acid dissolve with methanol solution and is settled to scale.
The preparation precision of 2.3 inner mark solutions takes in gaultherolin 27.84mg to 100mL measuring bottle, with E-C mixed liquor (2:1), dissolves and is diluted to scale.
Fruit of Chinese magnoliavine medicine materical crude slice powder (60 order) 0.2g is got in the preparation of 2.4 need testing solutions, accurately weighed, put in tool plug test tube, precision adds 10% sulfuric acid methyl alcohol 1.0mL solution, be placed in 35 ℃ of vibration 24h(150r/min of constant temperature oscillator) after, precision adds saturated NaCl solution 2mL and E-C mixed liquor (2:1) 2mL, DL 3min, centrifugal 5min (3000r/min), the accurate supernatant 1mL that draws, adds gaultherolin inner mark solution 1mL, adds appropriate anhydrous sodium sulfate, dry, obtain.
2.5GC-MS analysis condition
Mass spectrum condition: EI ion gun; Electron energy 70eV; 230 ℃ of ion source temperatures; 150 ℃ of level Four bar temperature; Detect voltage 1235V; Solvent delay 3.2min.
Chromatographic condition: HP-5 chromatographic column (μ m * 0.5,30m * 250 μ m); Helium (99.999%) is carrier gas, flow velocity 1.0mL/min; Before post, press 0.171MPa; 250 ℃ of injector temperatures; Split sampling (split ratio 15:1); Temperature programme: 40 ℃ of initial temperatures, rise to 140 ℃ with the speed of 5 ℃/min.
The investigation of 2.6 linear relationships
Accurate each 1mL of reference substance solution that draws is to tool plug test tube, according to " being placed in constant temperature oscillator " under preparation method's item of need testing solution, rise, operation in accordance with the law, carry out GC-MS analysis, take peak area as ordinate, and concentration is horizontal ordinate, drawing standard curve, regression equation is y=0.6297X-0.0192, r=0.9999.Experimental result shows that LA is good linear relationship within the scope of 0.514mg-3.104mg.Concrete outcome is shown in Fig. 4.
2.7 precision test
Get 0.794mg/mL4-oxopentanoic acid reference substance liquid METHOD FOR CONTINUOUS DETERMINATION 5 times, average peak area ratio is that 0.464, RSD is that 1.067% result shows that instrument precision is good.Concrete data result is in Table 2.
Table 2 GC-MS measures LA content precision test
2.8 stability test
The need testing solution of getting Chinese Magnolivine Fruit respectively 0,2,5,9,13,17,21h measures, average peak area ratio is that 0.409, RSD is 1.777%, result shows that sample is stable in 21h.Concrete data result is in Table 3.
Table 3 GC-MS measures the test of LA stable content
2.9 replica test
Parallel precision takes 5 parts of Chinese Magnolivine Fruit 0.2g, by operating under test sample item, makes need testing solution, carries out GC-MS mensuration.Average content is tried to achieve in calculating, and to be respectively 0.361%, RSD be 2.102%.Result shows that method repeatability is good.Concrete data result is in Table 4.
Table 4 GC-MS measures LA content replica test
2.10 average recovery test
Precision takes 3.69mg4-oxopentanoic acid standard items and puts in 5mL measuring bottle, adds 10%H
2sO
4methyl alcohol dissolves and is diluted to scale.Get Chinese Magnolivine Fruit 0.1g, accurately weighed, to put in tool plug test tube, precision adds the above-mentioned LA standard items of 0.5mL, then precision adds 10%H
2sO
4methyl alcohol 0.5mL, rises according to " being placed in constant temperature oscillator " under preparation method's item of need testing solution, and operation, carries out GC-MS analysis in accordance with the law.Calculating average recovery is that 99.762%, RSD is 2.640%.Concrete data result is in Table 5.
Table 5 GC-MS measures the test of LA content average recovery
Accurate reference substance and the need testing solution 1 μ l of drawing of 2.11 assays, inject gas chromatograph, measures (n=3), obtains.Concrete data result is in Table 6.Its concrete total ion current figure is shown in Fig. 5.
Table 6 GC-MS measures the content of LA in the fruit of Chinese magnoliavine
Claims (4)
1. a fruit of Chinese magnoliavine organic acid analysis method, is characterized in that comprising the following steps:
(1) sample preparation methods: get the about 0.2g of Chinese Magnolivine Fruit, accurately weighed, put in tool plug test tube, precision adds 10% sulfuric acid methyl alcohol 1.0mL in constant temperature oscillator after 35 ℃ of vibration 24h, and precision adds saturated NaCl solution 2mL and E-C mixed liquor 2mL, DL 3min, centrifugal 5min, gets supernatant, adds appropriate anhydrous sodium sulfate, dry, obtain;
(2) GC-M S analysis condition: mass spectrum condition: EI ion gun; Electron energy 70eV; 230 ℃ of ion source temperatures; 150 ℃ of level Four bar temperature; Detect voltage 1235V; Solvent delay 3.2min; Chromatographic condition: HP-5 chromatographic column; Helium is carrier gas, flow velocity 1.0mL/min; Before post, press 0.171MPa; 250 ℃ of injector temperatures; Split sampling; Temperature programme: 40 ℃ of initial temperatures, rise to 260 ℃ with the speed of 5 ℃/min, then keep 10min.
2. measure a method for LA content, it is characterized in that comprising the following steps:
(1) preparation of reference substance solution respectively the accurate LA reference substance of drawing put in right amount in 5mL measuring bottle, respectively add 10% sulfuric acid dissolve with methanol solution and be settled to scale;
(2) the preparation precision of inner mark solution takes gaultherolin in right amount to 100mL measuring bottle, with E-C mixed liquor, dissolves and is diluted to scale;
(3) Chinese Magnolivine Fruit 0.2g is got in the preparation of need testing solution, accurately weighed, puts in tool plug test tube, precision adds 10% sulfuric acid methyl alcohol 1.0mL solution, be placed in after 35 ℃ of vibration 24h of constant temperature oscillator, precision adds saturated NaCl solution 2mL and E-C mixed liquor 2mL, DL 3min, centrifugal 5min, the accurate supernatant 1mL that draws, adds gaultherolin inner mark solution 1mL, adds appropriate anhydrous sodium sulfate, dry, obtain;
(4) GC-MS analysis condition mass spectrum condition: EI ion gun; Electron energy 70eV; 230 ℃ of ion source temperatures; 150 ℃ of level Four bar temperature; Detect voltage 1235V; Solvent delay 3.2min.Chromatographic condition: HP-5 chromatographic column; Helium is carrier gas, flow velocity 1.0mL/min; Before post, press 0.171MPa; 250 ℃ of injector temperatures; Split sampling; Temperature programme: 40 ℃ of initial temperatures, rise to 140 ℃ with the speed of 5 ℃/min;
(5) accurate reference substance and the need testing solution 1 μ l of drawing of assay, inject gas chromatograph, measures, and obtains.
3. a fruit of Chinese magnoliavine method of quality control, is characterized in that, by the method for claim 2, measures LA content in the fruit of Chinese magnoliavine, and according to LA content meter in fruit of Chinese magnoliavine dry product, result is no less than setting value.
4. the content assaying method of LA according to claim 3, is characterized in that the setting value of the raw fruit of Chinese magnoliavine and steamed FRUCTUS SCHISANDRAE with vinegar is respectively 0.28%, 0.60% according to LA content meter in fruit of Chinese magnoliavine processed product dry product.
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| CN115236239A (en) * | 2022-08-09 | 2022-10-25 | 多特瑞(上海)商贸有限公司 | Method for detecting content of alpha-copal in schisandra essential oil |
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| CN115236239B (en) * | 2022-08-09 | 2023-12-29 | 多特瑞(上海)商贸有限公司 | Method for detecting content of alpha-copaene in schisandra essential oil |
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