CN106198791A - Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously - Google Patents

Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously Download PDF

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CN106198791A
CN106198791A CN201610506609.5A CN201610506609A CN106198791A CN 106198791 A CN106198791 A CN 106198791A CN 201610506609 A CN201610506609 A CN 201610506609A CN 106198791 A CN106198791 A CN 106198791A
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lignum aquilariae
aquilariae resinatum
benzylacetone
tetrol
sample
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CN106198791B (en
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李浩洋
杨芳
张宪臣
卢俊文
邓建
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INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY-EXIT INSPECTION AND QUARANTINE
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INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY-EXIT INSPECTION AND QUARANTINE
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation

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Abstract

The invention discloses a kind of Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in Lignum Aquilariae Resinatum of mensuration simultaneously, with methanol supersound process testing sample and do blank solution simultaneously, there is the hybrid standard working solution of Concentraton gradient with methanol preparation, wherein the concentration range of Lignum Aquilariae Resinatum tetrol is 20～200 μ g/mL, the concentration range of benzylacetone is 0.5～5 μ g/mL, utilize gas chromatograph-mass spectrometer to standard working solution, blank solution and sample solution are analyzed detection, obtain related color spectrogram, qualitative analysis is carried out with retention time and the characteristic ion abundance ratio of object in standard working solution with sample solution, quantitative analysis is carried out with external standard method, finally draw standard curve result of calculation.The present invention operates quickly, simple, and object can the advantage such as mensuration simultaneously, it is adaptable to Lignum Aquilariae Resinatum tetrol and the mensuration of benzylacetone in Lignum Aquilariae Resinatum.The inventive method is highly sensitive, reproducible, and the response rate is high.

Description

Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously
[technical field]
The present invention relates to a kind of Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in Lignum Aquilariae Resinatum of mensuration simultaneously, belong to detection method technology Field.
[background technology]
The timber that Lignum Aquilariae Resinatum is Isolated From Thymelaeaceae Species Lignum Aquilariae Resinatum or Lignum Aquilariae Resinatum contains resin, main product in China, Japan, India and its His country in Southeast Asia, has effect of promoting the circulation of QI to relieve pain, warming middle-JIAO to arrest vomiting, helping inspiration to relieve asthma, cures mainly chest and abdomen feeling of distension and oppression pain, gastrofrigid vomiting oh The diseases such as inverse, QI rising in reverse order of suffering from a deficiency of the kidney dyspnea with rapid respiration, are that China heals the main prescription raw medicinal material of hundred kinds of Chinese patent medicines.In addition to medicinal, Lignum Aquilariae Resinatum or name Expensive natural perfume material and incense raw material, the market demand is huge.But in nature, only it is upset when eaglewood or damages When hindering, just can produce Lignum Aquilariae Resinatum, Edgeworthia chrysantha Lindl. process generally requires the year the most up to a hundred decades, along with environmental destruction and Lignum Aquilariae Resinatum The over-exploitation of resource, eaglewood resource is the most exhausted, is now listed in country second class protection wild plant, and is loaded into " generation Conservation of nature alliance of boundary Endangered species Red List ", the demand in market can not be met the most far away, cause Lignum Aquilariae Resinatum price to be climbed year by year Rise.Presently commercially available Lignum Aquilariae Resinatum medical material is very different, there are substantial amounts of Lignum Aquilariae Resinatum inferior goods, even adulterant or Lignum Aquilariae Resinatum timber and soaks perfume Material, the counterfeit drug of utter misery post-treatment and dulcet tree material such as Cupressus funebris Endl. etc. fill generation, serious impact on medical effect.
Lignum Aquilariae Resinatum mainly contains chromone class, fragrance the composition such as the same clan and sesquiterpenoids, have document to 125 Lignum Aquilariae Resinatum product, its The agallochum material of his 25 kinds of dulcet tree materials and health detects, and result proves only chromone class just to be detected in Lignum Aquilariae Resinatum Composition Lignum Aquilariae Resinatum tetrol and aromatic component benzylacetone.It follows that Lignum Aquilariae Resinatum tetrol and benzylacetone are the characteristic components of Lignum Aquilariae Resinatum, Its content can be as Lignum Aquilariae Resinatum authenticity and the important evidence of quality evaluation.Therefore, version " Chinese Pharmacopoeia " regulation Lignum Aquilariae Resinatum in 2015 Middle Lignum Aquilariae Resinatum tetrol content is not less than 0.10%.
At present, the method for Lignum Aquilariae Resinatum tetrol and benzylacetone content in Lignum Aquilariae Resinatum that measures is respectively liquid chromatography and gas chromatogram Method, but there is no and measure the method for two kinds of compounds in Lignum Aquilariae Resinatum simultaneously.Although liquid chromatography and gas chromatography have stronger Separation analysis ability, but each constituent content in the mensuration product that they can only be quantitative, for qualitative identification quickly and accurately but Having some limitations, and use liquid-phase chromatographic analysis solvent consumption big, testing cost is high.The present invention use gas chromatogram- Tandem mass spectrum method, it is possible to measure Lignum Aquilariae Resinatum tetrol and benzylacetone content in Lignum Aquilariae Resinatum the most accurately, quickly and easily.
[summary of the invention]
The technical problem to be solved is to overcome the deficiencies in the prior art, it is provided that one can quickly, accurately calmly Property differentiate, Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in Lignum Aquilariae Resinatum can be measured again simultaneously.The inventive method uses gas chromatography-mass spectrum Combination detection method, has simple to operate, highly sensitive, reproducible, the advantage that target compound measures simultaneously.
The present invention for achieving the above object, by the following technical solutions:
Measuring instrument used in the present invention and equipment is: gas chromatograph-mass spectrometer (GC-MS), ultrasonic washing unit, electronics Balance.
Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously, it is characterised in that comprise the following steps:
A, prepare testing sample solution: cleaned by Lignum Aquilariae Resinatum sample ultra-pure water and dry, cross No. three sieves after pulverizing, accurately weigh 0.3g, in being placed in plug conical flask, is accurate to 0.0001g, the accurate methanol 12～15mL that adds, weighed weight, soaks 1h, frequency 40kHz, power 300W supersound process 50min, let cool, more weighed weight, supply the weight of loss with methanol, shake up, centrifugal, quiet Put, take the supernatant and i.e. obtain sample solution, to be measured, do blank solution simultaneously；
B, prepare standard working solution: there is the Lignum Aquilariae Resinatum tetrol of Concentraton gradient with methanol preparation and there is the benzyl of Concentraton gradient Benzylacetone hybrid standard working solution, wherein the concentration range of Lignum Aquilariae Resinatum tetrol is 20～200 μ g/mL, the concentration model of benzylacetone Enclosing is 0.5～5 μ g/mL；
C, gas chromatography-mass spectrometry analysis: utilize gas chromatograph-mass spectrometer (GC-MS) to standard working solution, blank solution and Sample solution is analyzed detection, obtains related color spectrogram, with in sample solution and standard working solution during the reservation of object Between and characteristic ion abundance ratio carry out qualitative analysis, carry out quantitative analysis with external standard method；
Wherein said gas chromatography-mass spectrum condition is:
Chromatographic column: HP-5MS capillary column, 30m × 0.25mm × 0.25 μm；Input mode: Splitless injecting samples, injection port Temperature: 250 DEG C；Carrier gas: He, 99.999%, post flow 1.2mL/min；Column temperature employing temperature programming: initial temperature 40 DEG C, protects Hold 1min, rise to 210 DEG C with 10 DEG C/min, keep 1min, then rise to 220 DEG C with 2 DEG C/min, keep 1min, then with 8 DEG C/min Rise to 300 DEG C, keep 4min；Sample size: 1 μ L；
Mass Spectrometry Conditions is: ion source temperature: 250 DEG C；Ionization pattern: electron impact ionization；Mass spectrum transmission line temperature 280 ℃；Solvent delay time 3.0min；
Scan mode: selected ion monitoring scanning (SIM), the monitoring period to each ion is 50ms；
D. draw standard curve and result calculates: standard working solution is introduced gas chromatograph-mass spectrometer (GC-MS), with external standard Method carries out quantitative analysis, the concentration of the peak area of target compound quota ion with target compound is carried out linear regression and divides Analysis, obtains standard curve equation of linear regression, and correlation coefficient need to be not less than 0.999；The sample solution of preparation is measured, surveys Obtain the peak area of target compound in sample solution, substitute into unary linear regression equation and obtain target compound in sample solution Content, converts further according to formula calculated below (1) and computing formula (2) and tries to achieve Lignum Aquilariae Resinatum tetrol and benzylacetone in sample respectively Content:
Computing formula (1) is:
X 1 = ( C - C 0 ) × V M × 10000 ... ( 1 )
Computing formula (2) is:
X 2 = ( C - C 0 ) × V M ... ( 2 )
In formula:
X1The content of Lignum Aquilariae Resinatum tetrol in sample, unit is %；
X2The content of benzylacetone in sample, unit is mg/kg；
The concentration of target compound in C sample solution, unit is μ g/mL；
C0The concentration of target compound in blank solution, unit is μ g/mL；
V sample solution constant volume, unit is milliliter (mL)；
The quality that M sample weighs, unit is g.
When calculating according to formula, being final measurement result with the meansigma methods of twice parallel determinations, result retains 3 to be had Effect three numeral.
The targeted Lignum Aquilariae Resinatum of the present invention includes the Lignum Aquilariae Resinatum that import Lignum Aquilariae Resinatum, Home-made occluder and employing artificial Edgeworthia chrysantha Lindl. technology produces.
The present invention is when preparing standard working solution, and the gradient concentration of Lignum Aquilariae Resinatum tetrol is 20 μ g/mL, 40 μ g/mL, 80 μ g/ ML, 120 μ g/mL, 160 μ g/mL, 200 μ g/mL, the gradient concentration of benzylacetone is 0.5 μ g/mL, 1 μ g/mL, 2 μ g/mL, 3 μ g/ mL、4μg/mL、5μg/mL.Concrete preparation method is:
Accurately weigh 0.1g benzylacetone standard substance, be accurate to 0.1mg, be placed in 100mL volumetric flask, dissolve also with methanol Constant volume, is configured to the benzylacetone standard reserving solution I that concentration is 1mg/mL；Accurately weigh 0.02g Lignum Aquilariae Resinatum tetrol standard substance, accurately To 0.1mg, it is placed in 10mL volumetric flask, dissolves with methanol, accurately pipette 0.500mL benzylacetone standard reserving solution I simultaneously and put In same 10mL volumetric flask and constant volume, it is configured to hybrid standard storing solution II, wherein Lignum Aquilariae Resinatum tetrol and the concentration of benzylacetone It is respectively 2mg/mL and 50 μ g/mL；Accurately pipette the hybrid standard storage of 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL Standby liquid II, is respectively placed in 10mL volumetric flask, with methanol constant volume to scale, obtains standard working solution.
In the present invention, during selected ion monitoring scanning, Lignum Aquilariae Resinatum tetrol and benzylacetone ion quantitative, qualitative and abundance thereof Than see table 1:
Table 1: Lignum Aquilariae Resinatum tetrol and benzylacetone quantitative and qualitative selection ion table
The present invention is drawing during standard curve and result calculate, the selection ion color of target compound and standard substance in sample Spectral peak (± 0.5%) at identical retention time occurs, and the mass-to-charge ratio of corresponding mass spectrometric fragment ion is consistent with standard substance, its Abundance ratio should meet compared with standard substance:
During relative abundance ＞ 50%, it is allowed to ± 10% deviation；
During relative abundance 20%～50%, it is allowed to ± 15% deviation；
During relative abundance 10%～20%, it is allowed to ± 20% deviation；
During relative abundance≤10%, it is allowed to ± 50% deviation；Now can confirm target analytes.
The present invention compares ultrasonic and is heated to reflux two kinds of extracting modes, and result shows with methanol as Extraction solvent, ultrasonic The object content that 50min extracts is basically identical with the object content being heated to reflux 5h extraction.Ultrasonic extracting method is the highest Effect, and in normal-temperature operation, therefore supersound extraction mode can be selected.
The present invention select in the preparation process of testing sample solution methanol as Extraction solvent, with ethanol, acetone, dichloro Methane, ethyl acetate, acetonitrile, hexamethylene, absolute ether, petroleum ether are compared, and the extraction efficiency of methanol is optimum.Therefore preferably methanol is made For Extraction solvent.
The present invention has investigated the impact on measurement result of the sample broke degree, and result shows, after particle diameter is less than No. three sieves, to subtract Small particle, in sample, the measurement result of target compound is without notable change, and the relative standard deviation of measurement result is less than 5%, Sample homogeneity disclosure satisfy that the mensuration requirement of the present invention.Therefore, No. three sieves are crossed during the preferred sample pre-treatments of the present invention.
The present invention has investigated the solvent load impact on measurement result, result table in the preparation process of testing sample solution Bright in the case of solvent load is 12～15mL, extraction efficiency is preferable, is further added by solvent load, and extraction efficiency change is little. Therefore selecting solvent load from the point of view of cost-effective is 12～15mL.
The present invention has investigated the extraction time impact on measurement result, result table in the preparation process of testing sample solution Bright extraction time is more or less the same more than 50min, extraction efficiency.Therefore consider that the selective extraction time is from time-saving angle 50min。
The present invention have selected suitable standard working solution scope, it is adaptable to Lignum Aquilariae Resinatum tetrol and the survey of benzylacetone in Lignum Aquilariae Resinatum Fixed.
The present invention, during gas chromatography-mass spectrometry analysis, in order to obtain more preferably separating effect, and avoids other group Divide and benzylacetone and Lignum Aquilariae Resinatum tetrol interfered, through repeatedly debugging optimization, heat up before have selected 210 DEG C and after 220 DEG C very fast, The slower heating schedule that heats up between 210 DEG C～220 DEG C detects.
In the present invention, when first passing through the reservation that full scan mode (SCAN) obtains benzylacetone and Lignum Aquilariae Resinatum tetrol standard substance Between and full scan spectrogram, then extract benzylacetone and the mass spectrum of Lignum Aquilariae Resinatum tetrol, and the phase of connexus spectrogram fragment ion respectively Pass information, determines the qualitative, quantitative ion of target compound.
Compared with prior art, the present invention has the following advantages:
The invention provides a kind of gas chromatography-mass spectrography and measure Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in Lignum Aquilariae Resinatum, This detection method has operation quickly, simple, and object can the advantage such as mensuration simultaneously, it is adaptable in Lignum Aquilariae Resinatum Lignum Aquilariae Resinatum tetrol and The mensuration of benzylacetone.
Extracting mode, Extraction solvent, sample broke degree, solvent, in order to obtain suitable Pretreatment, are used by the present invention Amount and extraction time are optimized and select, and employing can provide qualitative and quantitative information gas chromatography-mass spectrography simultaneously Lignum Aquilariae Resinatum tetrol in Lignum Aquilariae Resinatum sample and benzylacetone, as detecting instrument, are measured by instrument (GC/MS), and result shows that standard is bent Line linear dependence is good, and the detection of Lignum Aquilariae Resinatum tetrol is limited to 80mg/kg, and relative standard deviation is 3.47%, average recovery of standard addition Between 92.6%～97.5%, the detection of benzylacetone is limited to 2mg/kg, and relative standard deviation is 4.04%, and average mark-on returns Yield between 94.7%～103%, this shows that this method is highly sensitive, reproducible, and the response rate is high.
[accompanying drawing explanation]
Fig. 1 is the chromatogram of standard working solution；
Fig. 2 is the chromatogram of blank solution；
Fig. 3 is the chromatogram of sample solution；
Fig. 4 is the full scan chromatogram of standard working solution；
Fig. 5 is Lignum Aquilariae Resinatum four alcohol extraction ion massspectrum figure；
Fig. 6 is that benzylacetone extracts ion massspectrum figure.
[detailed description of the invention]
Below in conjunction with detailed description of the invention, the present invention done further more detailed description.
Embodiment 1:
Key instrument and reagent:
TSQ Quantum XLS Series gas chromatograph-GC-MS and work station (match Mo Feishier company of the U.S.) thereof； UA22MFD ultrasonic washing unit (WIGGENS company of China)；SIGMA 2-16K desk centrifuge (Germany sartoriussigma Company)；CP 225D type electronic balance (sensibility reciprocal: 0.0001g, Beijing Sai Duolisi company).
Lignum Aquilariae Resinatum tetrol standard substance (96.8%, National Institute for Food and Drugs Control), benzylacetone standard substance (98.0%, Aladdin reagent (Shanghai) Co., Ltd.), methanol, ethanol, acetone, dichloromethane, ethyl acetate, acetonitrile, hexamethylene, anhydrous second Ether, petroleum ether (chromatograph alcohol, Merck company of Germany).Useful water is ultra-pure water (resistivity is less than 18.2 μ s/cm).
Prepare testing sample solution:
Lignum Aquilariae Resinatum sample ultra-pure water is cleaned and dries, after pulverizing, cross No. three sieves, accurately weigh 0.3g and be placed in tool plug conical flask In, it is accurate to 0.0001g.The accurate methanol 15mL that adds, weighed weight, soak 1h, supersound process (frequency 40kHz, power 300W) 50min, lets cool, more weighed weight, supplies the weight of loss with methanol, shakes up, centrifugal, stands, takes the supernatant and get final product Sample solution, to be measured, do blank assay simultaneously.
Prepare standard working solution:
Accurately weigh 0.1020g benzylacetone standard substance, be placed in 100mL volumetric flask, dissolve and constant volume with methanol, preparation The benzylacetone standard reserving solution I becoming concentration to be 1mg/mL；Accurately weigh 0.0207g Lignum Aquilariae Resinatum tetrol standard substance, be placed in 10mL and hold In measuring bottle, dissolve with methanol, accurately pipette 0.500mL benzylacetone standard reserving solution I simultaneously and be placed in same 10mL volumetric flask And constant volume, it is configured to hybrid standard storing solution II, wherein the concentration of Lignum Aquilariae Resinatum tetrol and benzylacetone is respectively 2mg/mL and 50 μ g/ mL；Accurately pipette the hybrid standard storing solution II of 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, be placed in 10mL capacity In Ping, with methanol constant volume to scale, obtain standard working solution, wherein the gradient concentration of Lignum Aquilariae Resinatum tetrol be 20 μ g/mL, 40 μ g/ ML, 80 μ g/mL, 120 μ g/mL, 160 μ g/mL, 200 μ g/mL, the gradient concentration of benzylacetone is 0.5 μ g/mL, 1 μ g/mL, 2 μ g/mL、3μg/mL、4μg/mL、5μg/mL。
Gas chromatography-mass spectrometry analysis:
Utilize gas chromatograph-mass spectrometer (GC-MS) that standard working solution, blank solution and sample solution are analyzed detection, Obtaining related color spectrogram, see Fig. 1, Fig. 2 and Fig. 3, in figure, abscissa represents that retention time, vertical coordinate represent response value.With sample Solution carries out qualitative analysis with retention time and the characteristic ion abundance ratio of object in standard working solution, carries out with external standard method Quantitative analysis, described gas chromatography-mass spectrum condition is:
Chromatographic column: HP-5MS capillary column, 30m × 0.25mm × 0.25 μm；Input mode: Splitless injecting samples, injection port Temperature: 250 DEG C；Carrier gas: He, 99.999%, post flow 1.2mL/min；Column temperature employing temperature programming: initial temperature 40 DEG C, protects Hold 1min, rise to 210 DEG C with 10 DEG C/min, keep 1min, then rise to 220 DEG C with 2 DEG C/min, keep 1min, then with 8 DEG C/min Rise to 300 DEG C, keep 4min；Sample size: 1 μ L；
Mass Spectrometry Conditions is: ion source temperature: 250 DEG C；Ionization pattern: electron impact ionization；Mass spectrum transmission line temperature 280 ℃；Solvent delay time 3.0min；
Scan mode is that Salbutamol Selected Ion Monitoring scans (SIM), wherein Lignum Aquilariae Resinatum tetrol and benzylacetone ion quantitative, qualitative and Its abundance ratio is shown in Table 1, and the monitoring period to each ion is 50ms.
Drafting standard curve result of calculation:
Standard working solution is introduced gas chromatograph-mass spectrometer (GC-MS), carries out quantitative analysis with external standard method, by target chemical combination The peak area of thing and the concentration of target compound carry out linear regression analysis, obtain standard curve equation of linear regression, phase relation Number need to be not less than 0.999；The sample solution of preparation is measured, records the peak area of target compound, generation in sample solution Enter unary linear regression equation and obtain the content of target compound in sample solution, according to formula calculated below (1) and calculating public affairs Formula (2) converts and tries to achieve Lignum Aquilariae Resinatum tetrol and the content of benzylacetone in sample respectively:
Computing formula (1) is:
X 1 = ( C - C 0 ) × V M × 10000 ... ( 1 )
Computing formula (2) is:
X 2 = ( C - C 0 ) × V M ... ( 2 )
In formula:
X1The content of Lignum Aquilariae Resinatum tetrol in sample, unit is %；
X2The content of benzylacetone in sample, unit is mg/kg；
The concentration of target compound in C sample solution, unit is μ g/mL；
C0The concentration of target compound in blank solution, unit is μ g/mL；
V sample solution constant volume, unit is mL；
The quality that M sample weighs, unit is g；
Being final measurement result with the meansigma methods of twice parallel determinations, result retains 3 effective three numerals.
The range of linearity of the inventive method, detection limit and quantitative limit:
Standard working solution is introduced gas chromatograph-mass spectrometer (GC-MS), carries out quantitative analysis with external standard method, by target chemical combination The peak area of thing and the concentration of target compound carry out linear regression analysis, obtain the standard curve range of linearity, regression equation, phase Close coefficient, further accordance with the spectrogram of measured portions ion each after standard working solution sample introduction, in combination with sample solution spectrogram, then with Three times of signal to noise ratios (S/N=3) calculate detection limits, are computed assessing and draw benzylacetone and the detection limit of Lignum Aquilariae Resinatum tetrol.Result is shown in Table 2.
Table 2: the range of linearity, regression equation, correlation coefficient and detection limit
The inventive method precision and recovery test:
Parallel 7 parts of the Lignum Aquilariae Resinatum sample that weighs, carries out 7 independent trialss, the relative standard deviation of measurement result as stated above (RSD) being method precision, the RSD of Lignum Aquilariae Resinatum tetrol and benzylacetone is respectively 3.47% and 4.04%.The most parallel weighing can 9 parts of Lignum Aquilariae Resinatum sample, carries out the mark-on recovery test of high, medium and low 3 concentration levels, the average recovery rate of target compound respectively Between 92.6%～103%, its result is listed in table 3.
Table 3: method precision and the response rate
The optimization of Pretreatment and selection:
One, in order to obtain suitable Pretreatment, to extracting mode, Extraction solvent, sample broke degree, solvent load and Extraction time is optimized and selects.
1. the optimization of extracting mode and selection:
The present invention compares ultrasonic and is heated to reflux two kinds of extracting modes, and result shows with methanol as Extraction solvent, ultrasonic The target compound content that 50min extracts is basically identical with the object measured value being heated to reflux 5h extraction.Because of supersound extraction side Method rapidly and efficiently, and in normal-temperature operation, therefore can select supersound extraction mode, refers to table 4.
Table 4: the optimization of extracting mode and selection
2. the optimization of Extraction solvent and selection:
The present invention is respectively with methanol, ethanol, acetone, dichloromethane, ethyl acetate, acetonitrile, hexamethylene, absolute ether and stone Sample, as Extraction solvent, is detected by oil ether.Measurement result shows, methanol, ethanol, dichloromethane and ethyl acetate are to heavy The extraction efficiency of fragrant tetrol apparently higher than acetone, acetonitrile, hexamethylene, absolute ether and petroleum ether, and when select methanol, ethanol, When dichloromethane and ethyl acetate are as Extraction solvent, the measurement result no significant difference of Lignum Aquilariae Resinatum tetrol；Methanol and acetone are to benzyl The extraction efficiency of benzylacetone apparently higher than ethanol, dichloromethane, ethyl acetate, acetonitrile, hexamethylene, absolute ether and petroleum ether, And when selecting methanol and acetone as Extraction solvent, the measurement result no significant difference of benzylacetone.It is therefore preferable that methanol is made Extraction solvent for the present invention.Refer to table 5.
Table 5: the optimization of Extraction solvent and selection
Note:: " ND " represents that target compound does not detects.
3. the optimization of sample broke degree and selection:
By Lignum Aquilariae Resinatum sample comminution, after crossing a sieve, No. two sieves, No. three sieves, No. four sieves respectively, examine as stated above Survey.As known from Table 6, when particle diameter is more than No. three sieves, the relative standard deviation (RSD) of measurement result is excessive, the weight of testing result Renaturation can not meet the mensuration requirement of the present invention, and when particle diameter is less than No. three sieves, sample homogeneity increases, measurement result RSD is less than 5%, it is possible to meet the mensuration requirement of the present invention.When particle diameter is less than No. three sieves, after crossing No. three sieves, No. four sieves Sample is measured, the measurement result no significant difference of target compound.Therefore, No. three are crossed during the preferred sample pre-treatments of the present invention Sieve.
Table 6: the optimization of sample comminution degree and selection
4. the optimization of solvent load and selection:
The present invention in Lignum Aquilariae Resinatum sample respectively accurate add methanol 6,9,12,15,18mL detects.Measurement result table Bright, after methanol usage is more than 9mL, increase methanol usage, the measurement result of Lignum Aquilariae Resinatum tetrol still has an increase, and the survey of benzylacetone Determine result without significant change；After methanol usage is more than 12mL, increase methanol usage, the mensuration knot of Lignum Aquilariae Resinatum tetrol and benzylacetone Fruit has no significant change, therefore from the point of view of cost-effective, selecting solvent load is 12～15mL.Refer to table 7.
Table 7: the optimization of solvent load and selection
5. the optimization of extraction time and selection:
The present invention in the preparation process of testing sample solution, the most ultrasonic 30,40,50,60,70min detects.Survey Determine result to show, upon extracting between more than 40min after, increase extraction time, the measurement result of Lignum Aquilariae Resinatum tetrol still has an increase, and benzyl The measurement result of benzylacetone is without significant change；Between upon extracting more than 50min after, increase extraction time, Lignum Aquilariae Resinatum tetrol and benzyl The measurement result of acetone has no significant change, therefore considers from time-saving angle, and the selective extraction time is 50min.Refer to table 8。
Table 8: the optimization of extraction time and selection
Two, gas chromatogram--the optimization of mass spectrometry instrument parameter and selection:
In order to obtain suitable gas chromatogram--mass spectrometry instrument parameter, to heating schedule, qualitative ion and quota ion It is optimized and selects.
1. heating schedule optimization:
During actual sample detection, when programming rate is very fast, about 210 DEG C have a lot of aromatic components and sesquialter note component Concentrate out peak, retention time to be closer to, in order to obtain more preferably separating effect, and avoid other component to Lignum Aquilariae Resinatum tetrol and benzyl Benzylacetone interferes, and through repeatedly debugging optimization, heats up very fast before the present invention have selected 210 DEG C and after 220 DEG C, 210 DEG C～220 The slower heating schedule that heats up between DEG C detects.
2. the optimization of qualitative, quantitative ion and selection:
In the present invention, first pass through full scan mode (SCAN) and obtain Lignum Aquilariae Resinatum tetrol and the full scan spectrogram of benzylacetone, Seeing Fig. 4, in figure, abscissa represents that retention time, vertical coordinate represent response value；Extract benzylacetone and Lignum Aquilariae Resinatum tetrol the most respectively Mass spectrum, is shown in Fig. 5 and Fig. 6 respectively, and in figure, abscissa represents that mass number, vertical coordinate represent abundance.According to the fragment in mass spectrum Ion selection abundance is of a relatively high, mass number is relatively big and disturbs few characteristic ion fragment, finally determines target compound Qualitative, quantitative ion, is shown in Table 1.
Embodiment 2:
According to the assay method described in embodiment 1,10 batches of commercially available Lignum Aquilariae Resinatum samples are detected, its Lignum Aquilariae Resinatum tetrol and benzyl The content of acetone is shown in Table 9.As shown in Table 9, the mark that in sample solution, the content of Lignum Aquilariae Resinatum tetrol and benzylacetone selects in the present invention In the range of quasi-working curve, standard working solution is Lignum Aquilariae Resinatum tetrol and the mensuration of benzylacetone be applicable to Lignum Aquilariae Resinatum.
Table 9:10 criticizes Lignum Aquilariae Resinatum tetrol and the content of benzylacetone in Lignum Aquilariae Resinatum sample

Claims (8)

1. one kind measures Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in Lignum Aquilariae Resinatum simultaneously, it is characterised in that comprise the following steps:
A, prepare testing sample solution: cleaned by Lignum Aquilariae Resinatum sample ultra-pure water and dry, cross No. three sieves after pulverizing, accurately weigh 0.3g It is placed in plug conical flask, has been accurate to 0.0001g, the accurate methanol 12～15mL that adds, weighed weight, soaks 1h, supersound process 50min, lets cool, more weighed weight, supplies the weight of loss with methanol, shakes up, centrifugal, stands, takes the supernatant and i.e. obtain sample Solution, to be measured, do blank solution simultaneously；
B, prepare standard working solution: there is the Lignum Aquilariae Resinatum tetrol of Concentraton gradient with methanol preparation and there is the benzyl third of Concentraton gradient Ketone hybrid standard working solution, wherein the concentration range of Lignum Aquilariae Resinatum tetrol is 20～200 μ g/mL, and the concentration range of benzylacetone is 0.5～5 μ g/mL；
C, gas chromatography-mass spectrometry analysis: utilize gas chromatograph-mass spectrometer (GC-MS) to standard working solution, blank solution and sample Solution is analyzed detection, obtains related color spectrogram, with in sample solution and standard working solution object retention time and Characteristic ion abundance ratio carries out qualitative analysis, carries out quantitative analysis with external standard method；
Wherein said gas chromatography-mass spectrum condition is:
Chromatographic column: HP-5MS capillary column, 30m × 0.25mm × 0.25 μm；Input mode: Splitless injecting samples, injector temperature: 250℃；Carrier gas: He, 99.999%, post flow 1.2mL/min；Column temperature employing temperature programming: initial temperature 40 DEG C, keeps 1min, rises to 210 DEG C with 10 DEG C/min, keeps 1min, then rises to 220 DEG C with 2 DEG C/min, keeps 1min, then with 8 DEG C/min liter To 300 DEG C, keep 4min；Sample size: 1 μ L；
Mass Spectrometry Conditions is: ion source temperature: 250 DEG C；Ionization pattern: electron impact ionization；Mass spectrum transmission line temperature 280 DEG C；Molten Agent 3.0min time delay；
Scan mode: selected ion monitoring scans, and the monitoring period to each ion is 50ms；
D. draw standard curve and result calculates: standard working solution is introduced gas chromatograph-mass spectrometer (GC-MS), enters with external standard method Row quantitative analysis, carries out linear regression analysis by the concentration of the peak area of target compound quota ion Yu target compound, To standard curve equation of linear regression, correlation coefficient need to be not less than 0.999；The sample solution of preparation is measured, records sample The peak area of target compound in product solution, substitutes into unary linear regression equation and obtains containing of target compound in sample solution Amount, tries to achieve containing of Lignum Aquilariae Resinatum tetrol and benzylacetone in sample respectively further according to formula calculated below (1) and computing formula (2) conversion Amount:
Computing formula (1) is:
X 1 = ( C - C 0 ) × V M × 10000 ... ( 1 )
Computing formula (2) is:
X 2 = ( C - C 0 ) × V M ... ( 2 )
In formula:
X1The content of Lignum Aquilariae Resinatum tetrol in sample, unit is %；
X2The content of benzylacetone in sample, unit is mg/kg；
The concentration of target compound in C sample solution, unit is μ g/mL；
C0The concentration of target compound in blank solution, unit is μ g/mL；
V sample solution constant volume, unit is mL；
The quality that M sample weighs, unit is g.
Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously the most according to claim 1, its feature exists The Lignum Aquilariae Resinatum that import Lignum Aquilariae Resinatum, Home-made occluder and employing artificial Edgeworthia chrysantha Lindl. technology produces is included in described Lignum Aquilariae Resinatum.
Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously the most according to claim 1, its feature exists In step a, the condition that sample ultrasonic processes is frequency 40kHz, power 300W.
Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously the most according to claim 1, its feature exists In preparation standard working solution in, the gradient concentration of Lignum Aquilariae Resinatum tetrol is 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 120 μ g/mL, 160 μ g/mL, 200 μ g/mL, the gradient concentration of benzylacetone is 0.5 μ g/mL, 1 μ g/mL, 2 μ g/mL, 3 μ g/mL, 4 μ g/mL, 5 μ g/mL。
Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously the most according to claim 4, its feature exists In the method preparing standard working solution it is:
Accurately weigh 0.1g benzylacetone standard substance, be accurate to 0.1mg, be placed in 100mL volumetric flask, dissolve with methanol and determine Hold, be configured to the benzylacetone standard reserving solution I that concentration is 1mg/mL；Accurately weigh 0.02g Lignum Aquilariae Resinatum tetrol standard substance, be accurate to 0.1mg, is placed in 10mL volumetric flask, dissolves with methanol, accurately pipettes 0.500mL benzylacetone standard reserving solution I simultaneously and is placed in In same 10mL volumetric flask and constant volume, being configured to hybrid standard storing solution II, wherein the concentration of Lignum Aquilariae Resinatum tetrol and benzylacetone is divided Wei 2mg/mL and 50 μ g/mL；Accurately pipette the hybrid standard deposit of 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL Liquid II, is respectively placed in 10mL volumetric flask, with methanol constant volume to scale, obtains standard working solution.
Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously the most according to claim 1, its feature exists When selected ion monitoring scans, Lignum Aquilariae Resinatum tetrol and benzylacetone ion quantitative, qualitative and abundance ratio thereof see table:
Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously the most according to claim 1, its feature exists In drawing during standard curve and result calculate, in sample target compound with the selection chromatography of ions peak of standard substance identical guarantor Staying and occur at the time, and the mass-to-charge ratio of corresponding mass spectrometric fragment ion is consistent with standard substance, its abundance ratio should compared with standard substance Meet:
During relative abundance ＞ 50%, it is allowed to ± 10% deviation；
During relative abundance 20%～50%, it is allowed to ± 15% deviation；
During relative abundance 10%～20%, it is allowed to ± 20% deviation；
During relative abundance≤10%, it is allowed to ± 50% deviation；Now can confirm target analytes.
Lignum Aquilariae Resinatum tetrol and the method for benzylacetone in a kind of Lignum Aquilariae Resinatum of mensuration simultaneously the most according to claim 1, its feature exists In time calculating according to formula, being final measurement result with the meansigma methods of twice parallel determinations, result retains 3 effective three numbers Word.
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