CN109655543A - A method of identifying tuber of stemona medicine elementary source using UPLC characteristic spectrum - Google Patents
A method of identifying tuber of stemona medicine elementary source using UPLC characteristic spectrum Download PDFInfo
- Publication number
- CN109655543A CN109655543A CN201811569917.8A CN201811569917A CN109655543A CN 109655543 A CN109655543 A CN 109655543A CN 201811569917 A CN201811569917 A CN 201811569917A CN 109655543 A CN109655543 A CN 109655543A
- Authority
- CN
- China
- Prior art keywords
- peak
- medicinal material
- radix stemonae
- tuber
- stemona
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of methods for identifying tuber of stemona medicine elementary source using UPLC characteristic spectrum, the present invention first establishes the characteristic spectrum of tuber of stemona medicinal material, pass through comparative analysis, it is determined that identify the foundation of tuber of stemona medicine elementary source, this method is simple, reproducibility is good, accurately and reliably, the condition of the linear gradient elution of UPLC is simple, convenient for operation, it can be used for the identification of tuber of stemona medicine elementary source, the control for clinical raw material provides foundation.
Description
Technical field
The invention belongs to Pharmaceutical Analysis and technical field of medicine quality control, and in particular to a kind of to utilize UPLC characteristic spectrum
Identify the method for tuber of stemona medicine elementary source.
Background technique
The tuber of stemona is Stemonaceae plant radix stemonae sessilifoliae (StemonasessilifoliaMiq.), Radix stemonae japonicae
(StemonajaponicaMiq.) or the dried root of radix stemonae tuberosae (StemonatuberosaLour.).Spring, Qiu Erji are adopted
It digs, removes fibrous root, clean, set in boiling water and slightly scald or steam to without the white heart, take out, dry.The tuber of stemona has moistening lung to lower qi cough-relieving, desinsection
Effect.For the key medicine for treating tuberculosis, tubercle bacillus can be inhibited and kill consumptive disease worm, with it is basic absolutely, and can preventing phlegm from forming and stopping coughing, enriching yin
Moistening lung, lung yin is sufficient, and change source is advantageous, and lung is made to restore to declare respectful function, cures mainly more than, pulmonary tuberculosis of hundred coughs, chronic cough etc..According to Chinese Pharmacopoeia
2015 editions are recorded, and the tuber of stemona is common more base sources Chinese medicine, derive from Stemonaceae plant radix stemonae sessilifoliae Stemonasessilifolia
(Miq.) Miq., Radix stemonae japonicae Stemona japonica (Bl.) Miq. or radix stemonae tuberosae StemonatuberosaLour..It looks into
Lot of documents discovery is read, for the tuber of stemona because kind, the place of production are different, the type and content of main chemical compositions have certain difference.From
From the point of view of the distribution of structure type, in radix stemonae tuberosae based on I type and II type alkaloid, II type alkaloid is more in radix stemonae sessilifoliae, I
Type and IV type take second place, and II type and IV type alkaloid are more in Radix stemonae japonicae.In terms of bioactivity research, I, II, type III biology
Alkali has antitussive action, but best with I type.From literature research as a result, radix stemonae tuberosae kind is more excellent.
Literature research focuses primarily upon the chemical constitution study aspect of tuber of stemona medicinal material or medicine materical crude slice at present, and about tuber of stemona medicinal material
The research that Ji Yuan identifies is less.Therefore, it is necessary to provide a kind of tuber of stemona medicine elementary source mirror method for distinguishing.
Summary of the invention
In order to overcome the above-mentioned deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of utilization UPLC characteristic pattern
The method that spectrum identifies tuber of stemona medicine elementary source.
The present invention is achieved through the following technical solutions:
A method of identifying tuber of stemona medicine elementary source using UPLC characteristic spectrum, includes the following steps:
(a) preparation of test solution:
Radix stemonae tuberosae medicinal material and radix stemonae sessilifoliae medicinal material are taken respectively, it is finely ground, 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle,
It is appropriate that 50-100% methanol is added, is ultrasonically treated 10-30 minutes, takes out, lets cool, with 50-100% methanol constant volume to scale, shake
It is even, filtration to get；
(b) preparation of reference solution:
Take chlorogenic acid reference substance appropriate, add 80% methanol be made every 1ml containing 20 μ g of chlorogenic acid to get；Radix stemonae tuberosae is taken again
Control medicinal material, radix stemonae sessilifoliae control medicinal material 0.5g, it is accurately weighed, it sets in 20ml measuring bottle, it is appropriate to be added 80% methanol, ultrasonic treatment
30min, take out, let cool, with 80% methanol constant volume to scale, shake up, with 0.22um filter membrane filter to get；
(c) chromatographic condition and system suitability:
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as Mobile phase B,
According to the form below carries out gradient elution；Flow velocity: 0.25ml/min；Column temperature is 25 DEG C；Detection wavelength is 210nm；
(d) foundation of characteristic spectrum:
It is accurate respectively to draw reference solution and each 1 μ l of test solution, Ultra Performance Liquid Chromatography instrument is injected, measurement is adopted
It is matched with " similarity evaluation ", establishes the characteristic pattern of radix stemonae tuberosae medicinal material and radix stemonae sessilifoliae medicinal material
Spectrum；
(e) identify foundation:
The characteristic spectrum of radix stemonae tuberosae medicinal material and radix stemonae sessilifoliae medicinal material is compared, determination has in radix stemonae tuberosae medicinal material
8 characteristic peaks are referring to the peak peak S with 3 chlorogenic acid peak of peak, and the relative retention time of each characteristic peak is respectively as follows: peak 1:0.82, peak 2:
0.95, peak 3:1.00, peak 4:1.04, peak 5:1.18, peak 6:1.48, peak 7:1.52, peak 8:1.67, relative retention time are providing
Within ± the 10% of value；Determining in radix stemonae sessilifoliae medicinal material, there are 6 chromatographic peaks to lack peak 1 and peak compared with radix stemonae tuberosae medicinal material
2, by peak 1 and peak 2 as the foundation for identifying tuber of stemona medicine elementary source；
(f) identify:
Tuber of stemona sample to be measured is compared with radix stemonae tuberosae with the characteristic spectrum of radix stemonae sessilifoliae medicinal material, determines tuber of stemona medicinal material
Ji Yuan；If there is peak 1 and cutting edge of a knife or a sword 2, for radix stemonae tuberosae in tuber of stemona sample chromatogram to be measured；If without peak 1 and cutting edge of a knife or a sword 2, for radix stemonae sessilifoliae.
The technical solution further preferred as the present invention, it is described to identify tuber of stemona medicine elementary source using UPLC characteristic spectrum
Method is further included pointing out for step (g) peak 1 and peak 2: being measured using UPLC-Q-TOF/MS method,
Wherein, liquid-phase condition are as follows: using octadecylsilane chemically bonded silica as filler；Mobile phase: A:0.1% phosphoric acid；B:
Acetonitrile；Column temperature: 30 DEG C；Sample room temperature: 10 DEG C；Flow velocity: 0.30mL/min；Sampling volume: 2 μ L, negative ion mode；Mobile phase
Flow gradient see the table below:
Mass Spectrometry Conditions: atomization of the nitrogen as mass ion source, taper hole gas；Negative electrospray ionization mode；Capillary voltage:
2.5 KV；Orifice potential: 40 V；Extract orifice potential: 3 V；Ion source temperature: 100 DEG C；Desolvation temperature: 350 DEG C；Instead
To taper hole air-flow: 50 L/h；Desolvention gas velocity: 600 L/h；Collision gas flow velocity: 0.5mL/min；Sweep time: 0.5 s；It sweeps
Retouch time interval: 0.02 s；Mass charge ratio range: 50-1200 m/z；.
The present invention is pointed out by characteristic peak, and the peak 1 is attributed to neochlorogenic acid, and peak 2 is attributed to 3- coumaric acyl Kui
Peaceful acid.
This law is optimized by the preparation condition to test solution, to different solvents, different extracting mode,
Different extraction times are investigated, it is preferred that the preparation of step (a) test solution: taking radix stemonae tuberosae medicinal material and upright respectively
Tuber of stemona medicinal material, it is finely ground, 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle, 80% methanol of addition is appropriate, it is ultrasonically treated 10 minutes,
Take out, let cool, with 80% methanol constant volume to scale, shake up, filtration to get.
The technical solution further preferred as the present invention, a kind of utilization UPLC characteristic spectrum identify tuber of stemona medicinal material
The method of Ji Yuan further includes the assay of step (h) chlorogenic acid: using superelevation hplc determination, wherein
The preparation of test solution are as follows:
Tuber of stemona medicinal material is taken, it is finely ground, powder 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle, 70% ethyl alcohol of addition, at ultrasound
Reason 10 minutes, take out, let cool, be settled to scale with 70% ethyl alcohol, shake up, filter, take subsequent filtrate to get；
Chromatographic condition are as follows:
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as Mobile phase B,
Regulation according to the form below carries out gradient elution；Column temperature is 25 DEG C or less；Flow velocity is 0.25ml/min；Detection wavelength is 325nm；Reason
10000 should be not less than by calculating by the number of plates by chlorogenic acid；
Compared with prior art, the present invention having the following beneficial effects:
The present invention first establishes the characteristic spectrum of tuber of stemona medicinal material, passes through comparative analysis, it is determined that identifies tuber of stemona medicine elementary source
Foundation, this method is simple, and reproducibility is good, and accurately and reliably, the condition of the linear gradient elution of UPLC is simple, can convenient for operation
For the identification of tuber of stemona medicine elementary source, the control for clinical raw material provides foundation.
Detailed description of the invention
Fig. 1 is the stacking chart of 15 batches of radix stemonae tuberosae medicinal material characteristic spectrums；
Fig. 2 is that radix stemonae tuberosae and radix stemonae sessilifoliae medicinal material Ji Yuan identify figure；
Specific embodiment
Further illustrate that the present invention, following embodiment are the specific embodiment party of the present invention below by specific embodiment
Formula, but embodiments of the present invention are not limited by following embodiments.
1, instrument and reagent
Instrument be shown in Table 1, reagent be shown in Table 2 with reagent be shown in Table 3, medicinal material information is shown in Table 4.
1 device information summary sheet of table
2 reagent information summary sheet of table
3 reagent information summary sheet of table
4 15 batches of radix stemonae tuberosae medicinal material place of production information of table
Remarks: S1-S6 is field source.
2, the preparation of chromatographic condition and test solution
The preparation of 2.1 test solutions
Radix stemonae tuberosae medicinal material and radix stemonae sessilifoliae medicinal material are taken respectively, it is finely ground, 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle,
It is appropriate that 80% methanol is added, is ultrasonically treated 10 minutes, takes out, lets cool, with 80% methanol constant volume to scale, shake up, filters, i.e.,
?；The preparation of 2.2 reference solutions
Take chlorogenic acid reference substance appropriate, add 80% methanol be made every 1ml containing 20 μ g of chlorogenic acid to get；It is taken respectively to leaf again
Tuber of stemona control medicinal material, radix stemonae sessilifoliae control medicinal material 0.5g, it is accurately weighed, it sets in 20ml measuring bottle, it is appropriate to be added 80% methanol, ultrasound
(power 500W, frequency 40HKz) 30min is handled, is taken out, is let cool, with 80% methanol constant volume to scale, shake up, filtered with 0.22um
Membrane filtration cross to get.
2.3 chromatographic condition
Using octadecylsilane chemically bonded silica as filler, (column length 10cm, internal diameter 2.1mm, partial size are 1.6 μm；)；
Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as Mobile phase B, according to the form below regulation carries out gradient elution；Flow velocity: 0.25ml/min；
Column temperature is 25 DEG C；Detection wavelength is 210nm.
2.4 measuring method
It is accurate respectively to draw reference substance solution and each 1 μ l of test solution, inject superelevation liquid chromatograph, measurement.
3, sample solution preparation method is investigated
3.1, which extract solvent, investigates
Radix stemonae tuberosae medicinal material (lot number: S1) powder about 0.25g is taken, it is accurately weighed, it is separately added into 80% methanol, 50% first
Alcohol, methanol, water and appropriate amount of ethanol, ultrasonic treatment (power 500W, frequency 40kHz) 10 minutes, let cool, then fixed with corresponding solvent
Hold to scale, shake up, be centrifuged (8000rpm, 3min), supernatant filtration, take subsequent filtrate to get；According to determining under " 2.3 " item
Chromatographic condition, sample introduction record peak area, and the results are shown in Table 5.
5 radix stemonae tuberosae medicinal material characteristic spectrum different solvents of table are investigated
The result shows that chromatographic peak number is less when ethyl alcohol is as Extraction solvent, water, methanol, 50% methanol and 80% methanol
Chromatographic peak number and response it is all suitable, total peak area/sample weighting amount when 50% methanol and 80% methanol as solvent is larger, extracts
Efficiency is better than remaining 3 kinds of solvent, and total characteristic peak area/sample weighting amount value of 80% methanol is maximum, comprehensively considers final choice 80%
Methanol is Extraction solvent.
3.2 Extraction solvent modes are investigated
Take radix stemonae tuberosae medicinal material (lot number: S1) in right amount, it is finely ground, about 0.25g is taken, accurately weighed, parallel two parts, portion is placed in
In 10ml volumetric flask, 80% methanol of addition is appropriate, and ultrasonic treatment (power 500W, frequency 40kHz) 10 minutes is let cool, with 80%
Methanol constant volume is to graduation mark；80% methanol 10ml of a accurate addition, reflow treatment 10 minutes, lets cool, is supplied with 80% methanol
The weight of less loss, shakes up, filtration, take subsequent filtrate to get.
The results show that more and responded more preferably using the resulting chromatographic peak number of ultrasonic extraction mode, and simple to operation, therefore
This method selects ultrasonic treatment as test liquid extracting mode.
3.3 extraction times were investigated
Take radix stemonae tuberosae medicinal material (lot number: S1) in right amount, it is finely ground, about 0.25g is taken, it is accurately weighed, parallel three parts, set 10ml appearance
In measuring bottle, 80% methanol of addition is appropriate, and ultrasonic treatment (power 500W, frequency 45kHz) handles 10 minutes, 20 minutes, 30 minutes,
Let cool, respectively with 80% methanol constant volume to graduation mark, shake up, be centrifuged (8000 turns, 3 minutes), filtration, take subsequent filtrate to get；
The result shows that it is completely that experimental implementation is easy that ultrasonic extraction 10 minutes are i.e. extractable, final choice ultrasonic treatment 10
Minute.
4, chromatographic condition optimizes
The determination of 4.1 Detection wavelengths
It takes radix stemonae tuberosae medicinal material (lot number: S1), it is finely ground, about 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle, is added 80%
Appropriate methanol, ultrasonic (power 500W, frequency 40HKz) handle 10min, take out, let cool, with 80% methanol constant volume to scale, shake
It is even, it is filtered with 0.22um filter membrane to get the absorption spectrum within the scope of 190~400nm is recorded.
Experimental result: pass through mobile wavelength, at 210 nm, the chromatography that radix stemonae tuberosae medicinal material sample solution can detecte
Peak information is most wide, and the response of each chromatographic peak is larger, and Stemona alkaloids UV absorption is weak, most to absorb in 210nm, therefore selects
Selecting 210nm is Detection wavelength.
The selection of 4.2 mobile phases
Same test solution is taken, investigates -0.002% triethylamine solution of mobile phase acetonitrile, -0.1% phosphorus of acetonitrile respectively
Acid, -0.1% formic acid of acetonitrile are elution system, and by above-mentioned chromatographic condition, sample introduction is analyzed.
Experimental result, when -0.002% triethylamine solution of acetonitrile is as mobile phase, peak shape is poor, and baseline is unstable；Than
It is larger in the low wavelength interference such as 220nm with 0.1% formic acid compared with -0.1% formic acid of -0.1% phosphoric acid of acetonitrile and acetonitrile, with 0.1%
The chromatogram of phosphoric acid is ideal.Therefore selection -0.1% phosphoric acid system of acetonitrile.
5, methodological study
5.1 specificities are investigated
Precision draws reference solution, test solution, each 1 μ l of blank solvent, injects liquid chromatograph, presses " 2.3 " item
Lower chromatographic condition measurement.The results show that the analysis method can accurately detect pointed out characteristic peak, do not done by Extraction solvent
It disturbs, shows that the chromatographic condition has substantially met the maximum principle of information content.
5.2 precision are investigated
Radix stemonae tuberosae medicinal material (S1) is taken, is prepared into test liquid by sample solution preparation method, sample introduction 6 times, every time 1 μ l,
Investigate the consistency of characteristic peak relative retention time and relative peak area.As the result is shown: the RSD of each characteristic peak relative retention time
Maximum is 0.51%, and within the specified scope, instrument precision is good for each peak relative retention time and relative peak area.
5.3 repeatability are investigated
Totally 6 parts of same sample lots (lot number: S1) are taken, are prepared into test liquid, 1 μ l of sample introduction by sample solution preparation method
The consistency of characteristic peak relative retention time and relative peak area is investigated in analysis.As the result is shown: each characteristic peak relative retention time
Maximum is 0.75%, and within the specified scope, instrument repeatability is good for each peak relative retention time and relative peak area.
5.4 study on the stability
Take same sample lots (lot number: S1), be prepared into test liquid by sample solution preparation method, 0,2,4,6,8,
12,16,20,24 hours each sample introductions are primary, measure 24 hours altogether, respectively 1 μ l of sample introduction, investigate characteristic peak relative retention time and phase
To the consistency of peak area.As the result is shown: the RSD of the relative retention time of each characteristic peak is up to 0.69%, and each peak is opposite to be protected
Stay time and relative peak area within the specified scope, test liquid is good in 24 hours internal stabilities.
6, the foundation of radix stemonae tuberosae medicinal material characteristic spectrum
Radix stemonae tuberosae medicinal material 15 batches are taken, by test solution is prepared under " 2.1 " item, by the chromatostrip under " 2.3 " item
The measurement of part sample introduction, measurement result are shown in Table shown in 6,7." chromatographic fingerprints of Chinese materia medica phase is used to leaf medicinal material characteristic spectrum by 15 batches
Like degree evaluation software system " matching, generate characteristic spectrum stacking chart, the result is shown in Figure 1.
6 sample measurement result (relative retention time) of table
7 sample measurement result (relative peak area) of table
The results showed that 8 characteristic peaks should be presented in test sample characteristic spectrum, and should be with control medicinal material object of reference chromatography
In 8 characteristic peaks it is corresponding；Peak corresponding with chlorogenic acid object of reference peak is the peak S, calculates the opposite reservation at each characteristic peak and the peak S
Time, relative retention time should be within ± the 10% of specified value, it is specified that value are as follows: specified value are as follows: 0.82 (peak 1), 0.95 (peak
2), 1.04 (peaks 4), 1.18 (peaks 5), 1.48 (peaks 6), 1.52 (peaks 7), 1.67 (peaks 8)；It is opposite with peak 5 to calculate each characteristic peak
Peak area, relative peak area should be within the limits prescribed, it is specified that ranges are as follows: 0.451~3.559 (peak 1), 1.579~7.511
(peak 2), 0.420~2.649 (peak 3), 0.137~0.726 (peak 4), 1.808~16.747 (peaks 6), 0.421~4.460 (peak
7), 0.420~3.957 (peak 8).
7, identify foundation:
It establishes the characteristic spectrum of radix stemonae sessilifoliae according to the above method, determines that there are 6 chromatographic peaks in radix stemonae sessilifoliae medicinal material, and it is right
Leaf tuber of stemona medicinal material is compared, and peak 1 and peak 2 (such as Fig. 2) are lacked, by peak 1 and peak 2 as the foundation for identifying tuber of stemona medicine elementary source；Pass through
Characteristic peak is pointed out, and the peak 1 is attributed to neochlorogenic acid, and peak 2 is attributed to 3- coumaric acyl quininic acid.
8, Ji Yuan identifies:
Tuber of stemona sample to be measured is compared with radix stemonae tuberosae with the characteristic spectrum of radix stemonae sessilifoliae medicinal material, determines tuber of stemona medicinal material
Ji Yuan；If there is peak 1 and cutting edge of a knife or a sword 2, for radix stemonae tuberosae in tuber of stemona sample chromatogram to be measured；If without peak 1 and cutting edge of a knife or a sword 2, for radix stemonae sessilifoliae.
9, peak 1 and cutting edge of a knife or a sword 2 are pointed out: it is measured using UPLC-Q-TOF/MS method:
9.1 instruments and reagent
Instrument: SYNAPT G2 HDMS ultra high efficiency liquid phase flight time high resolution mass spectrum combined system (Waters
Corporation, Milford, MA, USA), data processing system be 4.1 work station of MarkerLynx (Waters,
Manchester, U.K.), AB135-S electronic analytical balance (Mei Tele-support benefit), KQ-118B sonic oscillation instrument (city of Kunshan
Ultrasonic instrument Co., Ltd).
Reagent: chromatography acetonitrile and methanol are purchased from J.T.Baker (Phillipsburg, NJ, USA), chromatographic grade formic acid, phosphorus
Acid, leucine enkephalin are purchased from Sigma Aldrich (MO, USA), experiment ultrapure water (18.2 M Ω) Milli-Q Water warfare
System prepares (Millipore, France), other agents useful for same are that analysis is pure.
9.2 experimental method
9.2.1 the preparation of test solution
Radix stemonae tuberosae medicinal material is taken, it is finely ground, 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle, 80% methanol of addition is appropriate, surpasses
Sonication 10 minutes, take out, let cool, with 80% methanol constant volume to scale, shake up, filtration to get；
9.2.2 analytical test strip part
9.2.2.1 liquid-phase condition: using Waters ACQUITY UPLC system (PDA detector), and configuration binary solvent is defeated
Send system and autosampler module.Chromatographic column: CORTECS UPLC T3 chromatographic column (100 mm × 2.1 mm, 1.6 μm)；Stream
Dynamic phase: A:0.1% phosphoric acid；B: acetonitrile；Column temperature: 30 DEG C；Sample room temperature: 10 DEG C；Flow velocity: 0.30mL/min；Sampling volume: 2 μ
L；Mobile phase flow gradient see the table below:
9.2.2.2 Mass Spectrometry Conditions: mass spectrum uses Waters SYNAPT G2 HDMS system.Nitrogen is as mass ion source
Atomization, taper hole gas；Electrospray ionisation negative ion mode；Capillary voltage: 2.5 KV；Orifice potential: 40 V；Extract taper hole electricity
Pressure: 3 V；Ion source temperature: 100 DEG C；Desolvation temperature: 350 DEG C；Reversed taper hole air-flow: 50 L/h；Desolvention gas velocity:
600 L/h；Collision gas flow velocity: 0.5mL/min；Sweep time: 0.5s；Trace interval: 0.02 s；Mass charge ratio range: 50-
1200 m/z；Data collection form: continuum；Sensitivity: normal；Dynamic range: extended.
9.2.2.3 experimental result: using the UPLC-Q-TOF/MS analysis method after optimization, radix stemonae tuberosae sample is carried out
Detection.It is compared by the chromatographic peak retention behavior of compound, accurate molecular weight and MSE fragment information and standard items, to spy
It is pointed out at sign peak.
The identification at peak 1:
Under negative ion mode, peak 1 provides m/z 353.0898 in first mass spectrometric figure, is quasi-molecular ion peak [M-H]-
(C16H17O9Δm/z 7.0ppm)；In second order ms, the biggish fragment ion peak m/z 191,179 and 135 of abundance is produced；
In addition, there are the characteristic peaks of 240,280 and 320 nm in the ultraviolet spectra of the chromatographic peak.It is compared by consulting literatures, is tentatively pushed away
Break as neochlorogenic acid, is compared finally by with standard items, determine that it is neochlorogenic acid (Neochlorogenic acid).
The identification at peak 2:
Under negative ion mode, peak 2 provides m/z 367.1049 in first mass spectrometric figure, is quasi-molecular ion peak [M-H]-
(C17H19O9Δm/z 5.4ppm)；In second order ms, fragment ion, respectively m/z 193 (100) are produced, 191,173,
134；In addition, there is the characteristic peak of 240 and 320 nm in the ultraviolet spectra of the chromatographic peak.It is compared by consulting literatures, tentatively
It is inferred as feruloylquinic acid class compound.Further according to its mass spectrographic base peak ion, discovery 2 fragment ion base peak of peak is m/z
193 (100), thus it is speculated that it may be 3-FQA (3-feruloylquinic acid).
10, determination of chlorogenic acid
10.1, the preparation of chromatographic condition and test solution
10.1.1 the preparation of test solution
Take radix stemonae tuberosae medicinal material appropriate, it is finely ground, powder 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle, 70% second is added
Appropriate alcohol, ultrasonic treatment (500W, 40kHz) 10 minutes are taken out, let cool, be settled to scale with 70% ethyl alcohol, shake up, and filter, take
Subsequent filtrate to get.
10.1.2 the preparation of reference solution
Take chlorogenic acid reference substance appropriate, it is accurately weighed, add methanol that the stock solution of every 1ml 0.5mg containing chlorogenic acid is made；Essence
It is close to pipette above-mentioned stock solution 1ml, with methanol dilution at the reference substance solution of every 1ml 0.025mg containing chlorogenic acid.
10.1.3 chromatographic condition
Chromatographic condition is as follows: using octadecylsilane chemically bonded silica as filler (column length 10cm, internal diameter 2.1mm, grain
Diameter is 1.7 μm)；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as Mobile phase B, the regulation according to the form below carries out gradient elution；Column
Temperature is 25 DEG C or less；Flow velocity is 0.25ml/min；Detection wavelength is 325nm.Theoretical cam curve should be not less than by chlorogenic acid calculating
10000。
10.1.4 measuring method
It is accurate respectively to draw reference substance solution and each 1 μ L of test solution, inject superelevation liquid chromatograph, measurement.
10.2, methodological study
10.2.1 accuracy
Known content radix stemonae tuberosae medicinal material (lot number: S5) about 0.125g is taken, accurately weighed, every batch of is weighed 6 parts parallel, often
The chlorogenic acid reference substance of similar amount is added in part, and by sample solution preparation method, every batch of prepares 6 parts of test solution.Every part
Precision is drawn 1 μ l and is measured by above-mentioned chromatographic condition, calculates the rate of recovery with following equation, RSD value the results are shown in Table 8.
8 sample recovery rate experimental result of table
Experimental result shows that the rate of recovery mean value of radix stemonae tuberosae medicinal material chlorogenic acid is 101.8%, RSD 2.2%, according to
" Chinese Pharmacopoeia " 2015 editions four " drug standard analysis method verification guide principle " provides component content to be measured in sample
When being 0.1%, rate of recovery limit is 90%~108%, shows that the rate of recovery meets the requirements.
10.2.2 precision
Precision draws test solution (lot number: S5), repeats sample introduction 6 times according to above-mentioned chromatographic condition, 1 μ l of sampling volume.
Peak area RSD is calculated, RSD 0.21%, the experimental results showed that instrument precision is good, measurement result is shown in Table 9.
9 precision of table investigates result
10.2.3 repeated
It takes with a collection of 6 parts of test solution (lot number: S5), every part of accurate 1 μ l that draws is measured by above-mentioned chromatographic condition, measurement
Test solution Content of Chlorogenic Acid calculates RSD, the experimental results showed that radix stemonae tuberosae medicinal material content results RSD < 3%, the analysis
The repeatability of method is good, and measurement result is shown in Table 10.
10 repetitive test of table
10.2.4 specificity
Precision draws test solution (lot number: S5), chlorogenic acid reference substance solution and each 1 μ l of blank solvent, injects liquid phase
Chromatograph carries out 210nm~400nm Scanning Detction according to above-mentioned chromatographic condition with PDA detector.It the results are shown in Table 11-12.
11 chromatographic peak separation parameter of table
12 chromatographic peak purity parameter of table
The experimental results showed that there is no chromatographic peak in retention time corresponding with chlorogenic acid in blank solvent chromatography, illustrate solvent
It is noiseless to the measurement of chlorogenic acid；Impurity peaks are not detected in sample Content of Chlorogenic Acid, and the peak purity factor is in calculated threshold limit
It is interior, illustrate under the chromatographic condition, chlorogenic acid peak purity meets the requirements.In conclusion containing measurer with this law measurement chlorogenic acid
There is specificity.
10.2.5 linear
Precision pipettes that reference substance stock solution is appropriate, is shaken up, is pressed at the reference substance solution of series of concentrations with methanol dilution respectively
Above-mentioned chromatographic condition successively 1 μ l of sample introduction records peak area, as a result such as table 13:
13 chlorogenic acid of table linearly investigates result
Equation of linear regression are as follows: y=12496342.3453x-1903.8887, correlation coefficient r2=1.0000, show green
Ortho acid linear relationship in the 5.47 μ g/ml concentration ranges of μ g/ml~43.74 is good.
10.3 determination of chlorogenic acid
15 batches of radix stemonae tuberosae medicinal materials are prepared into test solution according to the method described above, content is measured, the results are shown in Table 14.
The chlorogenic acid content result of 14 15 batches of radix stemonae tuberosae medicinal materials of table
Claims (5)
1. a kind of method for identifying tuber of stemona medicine elementary source using UPLC characteristic spectrum, which comprises the steps of:
(a) preparation of test solution:
Radix stemonae tuberosae medicinal material and radix stemonae sessilifoliae medicinal material are taken respectively, it is finely ground, 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle, is added
50-100% methanol is appropriate, is ultrasonically treated 10-30 minutes, takes out, lets cool, with 50-100% methanol constant volume to scale, shake up, and filters
Cross to get；
(b) preparation of reference solution:
Take chlorogenic acid reference substance appropriate, add 80% methanol be made every 1ml containing 20 μ g of chlorogenic acid to get；Take radix stemonae tuberosae pair again respectively
It is accurately weighed according to medicinal material, radix stemonae sessilifoliae control medicinal material 0.5g, it sets in 20ml measuring bottle, it is appropriate to be added 80% methanol, ultrasonic treatment
30min, take out, let cool, with 80% methanol constant volume to scale, shake up, with 0.22um filter membrane filter to get；
(c) chromatographic condition and system suitability:
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as Mobile phase B, according to the form below
Carry out gradient elution；Flow velocity: 0.25ml/min；Column temperature is 25 DEG C；Detection wavelength is 210nm；
(d) foundation of characteristic spectrum:
Accurate absorption reference solution and each 1 μ l of test solution respectively, injection Ultra Performance Liquid Chromatography instrument, measurement, using " in
Medicine chromatographic fingerprinting similarity evaluation system " matching, establishes the characteristic spectrum of radix stemonae tuberosae medicinal material and radix stemonae sessilifoliae medicinal material；
(e) identify foundation:
The characteristic spectrum of radix stemonae tuberosae medicinal material and radix stemonae sessilifoliae medicinal material is compared, determines that there are 8 in radix stemonae tuberosae medicinal material
Characteristic peak is referring to the peak peak S with 3 chlorogenic acid peak of peak, and the relative retention time of each characteristic peak is respectively as follows: peak 1:0.82, peak 2:
0.95, peak 3:1.00, peak 4:1.04, peak 5:1.18, peak 6:1.48, peak 7:1.52, peak 8:1.67, relative retention time are providing
Within ± the 10% of value；Determining in radix stemonae sessilifoliae medicinal material, there are 6 chromatographic peaks to lack peak 1 and peak compared with radix stemonae tuberosae medicinal material
2, by peak 1 and peak 2 as the foundation for identifying tuber of stemona medicine elementary source；
(f) identify:
Tuber of stemona sample to be measured is compared with radix stemonae tuberosae with the characteristic spectrum of radix stemonae sessilifoliae medicinal material, determines tuber of stemona medicinal material base
Source；If there is peak 1 and cutting edge of a knife or a sword 2, for radix stemonae tuberosae in tuber of stemona sample chromatogram to be measured；If without peak 1 and cutting edge of a knife or a sword 2, for radix stemonae sessilifoliae.
2. a kind of method for being identified tuber of stemona medicine elementary source using UPLC characteristic spectrum according to claim 1, feature are existed
In, further include pointing out for step (g) peak 1 and peak 2: being measured using UPLC-Q-TOF/MS method,
Wherein, liquid-phase condition are as follows: using octadecylsilane chemically bonded silica as filler；Mobile phase: A:0.1% phosphoric acid；B: acetonitrile；
Column temperature: 30 °C；Sample room temperature: 10 °C；Flow velocity: 0.30mL/min；Sampling volume: 2 μ L, negative ion mode；Mobile phase stream
Dynamic gradient see the table below:
Mass Spectrometry Conditions: atomization of the nitrogen as mass ion source, taper hole gas；Electrospray ionisation negative ion mode；Capillary voltage:
2.5 KV；Orifice potential: 40 V；Extract orifice potential: 3 V；Ion source temperature: 100 °C；Desolvation temperature: 350 °C；Instead
To taper hole air-flow: 50 L/h；Desolvention gas velocity: 600 L/h；Collision gas flow velocity: 0.5mL/min；Sweep time: 0.5s；It sweeps
Retouch time interval: 0.02 s；Mass charge ratio range: 50-1200 m/z.
3. a kind of method for being identified tuber of stemona medicine elementary source using UPLC characteristic spectrum according to claim 2, feature are existed
In,
The peak 1 is attributed to neochlorogenic acid, and peak 2 is attributed to 3- coumaric acyl quininic acid.
4. a kind of method for being identified tuber of stemona medicine elementary source using UPLC characteristic spectrum according to claim 1, feature are existed
In the preparation of step (a) test solution: radix stemonae tuberosae medicinal material and radix stemonae sessilifoliae medicinal material are taken respectively, it is finely ground, 0.25g is taken, essence
It is close weighed, it sets in 10ml measuring bottle, 80% methanol of addition is appropriate, is ultrasonically treated 10 minutes, takes out, lets cool, extremely with 80% methanol constant volume
Scale shakes up, filtration to get.
5. a kind of method for being identified tuber of stemona medicine elementary source using UPLC characteristic spectrum according to claim 1, feature are existed
In further including the assay of step (h) chlorogenic acid: using superelevation hplc determination, wherein
The preparation of test solution are as follows:
Tuber of stemona medicinal material is taken, it is finely ground, powder 0.25g is taken, it is accurately weighed, it sets in 10ml measuring bottle, 70% ethyl alcohol, ultrasonic treatment 10 is added
Minute, take out, let cool, be settled to scale with 70% ethyl alcohol, shake up, filter, take subsequent filtrate to get；
Chromatographic condition are as follows:
Using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% phosphoric acid as Mobile phase B, according to the form below
In regulation carry out gradient elution；Column temperature is 25 DEG C or less；Flow velocity is 0.25ml/min；Detection wavelength is 325nm；Theoretical tray
Number is calculated by chlorogenic acid should be not less than 10000；
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811569917.8A CN109655543B (en) | 2018-12-21 | 2018-12-21 | Method for identifying radix stemonae medicinal material source by using UPLC (ultra performance liquid chromatography) characteristic spectrum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811569917.8A CN109655543B (en) | 2018-12-21 | 2018-12-21 | Method for identifying radix stemonae medicinal material source by using UPLC (ultra performance liquid chromatography) characteristic spectrum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109655543A true CN109655543A (en) | 2019-04-19 |
| CN109655543B CN109655543B (en) | 2022-05-17 |
Family
ID=66116241
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811569917.8A Active CN109655543B (en) | 2018-12-21 | 2018-12-21 | Method for identifying radix stemonae medicinal material source by using UPLC (ultra performance liquid chromatography) characteristic spectrum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109655543B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012096463A2 (en) * | 2011-01-13 | 2012-07-19 | 대전대학교 산학협력단 | Composition containing a mixed herbal extract as an active ingredient for preventing and improving allergic or non-allergic skin disease, and uses thereof |
| CN107271598A (en) * | 2017-07-20 | 2017-10-20 | 青岛市食品药品检验研究院 | The construction method of Chinese patent drug Yanyan slice standard feature collection of illustrative plates and application |
| CN108426955A (en) * | 2018-03-11 | 2018-08-21 | 安徽省食品药品检验研究院 | The discrimination method of tuber of stemona genunie medicinal materials |
| CN108872410A (en) * | 2018-03-22 | 2018-11-23 | 滨州医学院 | A kind of method for building up and its finger-print of lung-nourishing semifluid extract finger-print |
-
2018
- 2018-12-21 CN CN201811569917.8A patent/CN109655543B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012096463A2 (en) * | 2011-01-13 | 2012-07-19 | 대전대학교 산학협력단 | Composition containing a mixed herbal extract as an active ingredient for preventing and improving allergic or non-allergic skin disease, and uses thereof |
| CN107271598A (en) * | 2017-07-20 | 2017-10-20 | 青岛市食品药品检验研究院 | The construction method of Chinese patent drug Yanyan slice standard feature collection of illustrative plates and application |
| CN108426955A (en) * | 2018-03-11 | 2018-08-21 | 安徽省食品药品检验研究院 | The discrimination method of tuber of stemona genunie medicinal materials |
| CN108872410A (en) * | 2018-03-22 | 2018-11-23 | 滨州医学院 | A kind of method for building up and its finger-print of lung-nourishing semifluid extract finger-print |
Non-Patent Citations (3)
| Title |
|---|
| LAN-LAN FAN 等: "Binary chromatographic fingerprint analysis of Stemonae Radix from three Stemona plants and its applications", 《JOURNAL OF NATURAL MEDICINES》 * |
| 吴燕 等: "UPLC测定川芎地上部分中的３种活性成分", 《华西药学杂志》 * |
| 董巍 等: "UPLC-Q-TOF-MS法分析对叶百部蜜炙前后化学成分的变化", 《中成药》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109655543B (en) | 2022-05-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Voyksner et al. | Optimization and application of particle beam high‐performance liquid chromatography/mass spectrometry to compounds of pharmaceutical interest | |
| CN105738546B (en) | Method for establishing fingerprint of radix curcumae medicinal material and fingerprint thereof | |
| CN104977370B (en) | A kind of LC-MS quickly detects the method for Pyrazine material in white wine | |
| CN109270187A (en) | A kind of Chinese materia medica preparation quality evaluating method based on metabolism group Yu full ingredient semi-quantitative analysis | |
| CN109212120A (en) | A kind of construction method and evodia rutaecarpa quality of medicinal material detection method of evodia rutaecarpa medicinal material characteristic spectrum | |
| CN108152399B (en) | Construction and detection method of UPLC (ultra performance liquid chromatography) characteristic spectrum of semen boitae medicinal material | |
| CN107064362B (en) | The measurement of parabens substance in buccal cigarette | |
| CN107529337A (en) | The HPLC analyses of impurity in two to the water wei ling alcohol | |
| CN109374785B (en) | Construction method and detection method of UPLC (ultra performance liquid chromatography) characteristic spectrum of lophatherum gracile medicinal material | |
| CN112557563A (en) | Method for identifying medicinal material fructus aurantii in western and Jiangxi regions | |
| Lee et al. | Effects of aerosol size and coating thickness on the molecular detection using extractive electrospray ionization | |
| CN109632995A (en) | A kind of method for building up of semen ziziphi spinosae flavones ingredient UPLC finger-print and application | |
| CN112858497B (en) | Method for constructing characteristic spectrum of rhizoma Menispermi standard decoction and detecting quality of rhizoma Menispermi standard decoction | |
| CN106018658A (en) | Multidimensional gas chromatography-tandem mass spectrometry analytical method for nicotine optical isomer in electronic cigarette smoke solution | |
| CN106918655A (en) | A kind of polygala UPLC assay methods | |
| CN113884597B (en) | HPLC-IT-TOF/MS fingerprint detection method for endive and fingerprint thereof | |
| CN109655543A (en) | A method of identifying tuber of stemona medicine elementary source using UPLC characteristic spectrum | |
| CN107576743A (en) | The method for building up and its finger-print of the peaceful spray finger-print of nose | |
| CN110487925A (en) | A kind of method of chemical component in quick identification lilii and Rehmanniae Decoction | |
| CN108205024B (en) | Fingerprint quality control method for ficus microcarpa dry extract | |
| CN109374787B (en) | Construction method and detection method of radix stemonae tuberose medicinal material UPLC characteristic spectrum | |
| CN114216980A (en) | Method for establishing HPLC-ELSD (high Performance liquid chromatography-evaporative light scattering) fingerprint spectrum of starwort root | |
| CN104062389B (en) | A kind of Folium camelliae assamicae GC finger printing test method of Different Extraction Method | |
| CN107976494B (en) | Construction of standard characteristic spectrum of Kangfu tincture and quality detection method thereof | |
| CN110361459B (en) | Method for establishing loquat leaf standard decoction fingerprint |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |