CN109307721A - A kind of HPLC-QQQ/MS method measures the detection method of content of effective component in Dragon groundnut - Google Patents
A kind of HPLC-QQQ/MS method measures the detection method of content of effective component in Dragon groundnut Download PDFInfo
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Abstract
The present invention provides the detection method of content of effective component in a kind of HPLC-QQQ/MS method measurement Dragon groundnut, wherein chromatographic condition are as follows: chromatographic column is using octadecyl silane column as filler, using 0.1% formic acid-water as mobile phase A, using 0.1% formic acid-acetonitrile as Mobile phase B, its volume proportion is mobile phase A phase: Mobile phase B phase are as follows: 80~0%:20~100% carries out gradient elution；This method can measure the Chinese medicine active ingredients ingredients such as Astragaloside IV in Dragon groundnut, hydroxyl radical carthamin yellow carthamus A, calycosin glucoside, calycosin, ferulic acid, Syringin, n butylphthalide, Ligustilide, Senkyunolide A, senkyunolide I, Senkynolide H, ligustrazine, isofraxidin, dehydro-α-curcumene, amarogentin, eleutheroside E, Paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin.The detection method of content has operating method simple, high sensitivity, the accurate advantage of content.
Description
Technical field
The present invention relates to belong to pcm chemical analysis detection field more particularly to a kind of HPLC-QQQ/MS method measurement dragon
The detection method of content of effective component in raw leech capsule.
Background technique
Dragon groundnut is produced without competition by Shaanxi Buchang Pharmaceuticals Co., Ltd., the drug be by Radix Astragali, leech, Rhizoma Chuanxiong,
Radix Angelicae Sinensis, peach kernel, safflower, radix paeoniae rubra, radix aucklandiae, rhizoma acori graminei, 12 taste Chinese medicinal composition of pheretima, herba taxilli and wilsonii compound preparation, tool
Have inrigorating qi and promoting blood circulation, by blood stasis and smoothing collaterals the effect of.Clinical research shows that Dragon groundnut is reachable to cerebral infarction total effective rate before listing
91.7%, and to the common tcm syndrome improvement rate of apoplexy sequelae 80% or more.It is widely used in cerebral infarction after listing
The treatment of dead convalescence using Dragon groundnut or combines other application in TCM and obtains brilliance on the basis of western medicine
Effect can reduce answering for its state of an illness if the raw leech of use in conjunction dragon and aspirin for treatment transient ischemic attack are curative for effect
Hair rate and progression of the disease are the risk of cerebral apoplexy；Have on the basis of western medical treatment plus with Dragon groundnut treatment acute cerebral infarction
It improves significantly.
Relatively more to the clinical research of Dragon groundnut at present, clinical effectiveness is significant, but its chemical constitution study is still
It does not report, material base is still not clear, therefore carrying out assay to Dragon groundnut chemical component can be effectively to compound
Preparation carries out quality control, either guarantees that the safety and validity of medication, or accurate thoroughly evaluating quality of medicinal material are all
It is very necessary and significant.Document report is only using Astragaloside IV as the quality control index of Dragon groundnut at present, and Chinese medicine
Pharmacological effect is plurality of active ingredients synergistic effect as a result, the performance rating of preparation should be the investigation of Multiple components, is only passed through
Measuring single or several indexs more cannot comprehensively reflect traditional Chinese medicine quality, and multicomponent active constituent quantitative analysis is Chinese medicine matter
Amount controls most direct and necessary method.Furthermore high performance liquid chromatography-triplex tandem level four bars mass spectrography has quick, sensitive
High feature is spent, mass spectrum multiple-reaction monitoring scan pattern is determined according to the mass-to-charge ratio information of compound parent ion and daughter ion
Amount, can be improved detection sensitivity and dosing accuracy, be to solve the quantitative powerful of Multi-chemical ingredients.Therefore, compel
It is essential and wants a kind of easy, efficient, accurate, at low cost quality determining method energy qualitative simultaneously and quantitative determination Dragon groundnut
In multiple active principles.
Summary of the invention
The object of the present invention is to provide the content detections of effective component in a kind of HPLC-QQQ/MS method measurement Dragon groundnut
Method, the detection method is to 19 kinds of effective components of the medicinal materials such as Radix Astragali, Rhizoma Chuanxiong in Dragon groundnut, using multiple-reaction monitoring mould
Formula carries out component content measurement to 3 batches of commercially available Dragon groundnuts, carries out detection point to the active principle component content in the raw leech of dragon
Analysis provides scientific basis to control for the quality of Dragon groundnut and evaluate its interior quality.
Dragon groundnut of the present invention, each means and is produced by Shaanxi Buchang Pharmaceuticals Co., Ltd., and the drug is by Huang
225 parts of stilbene, 100 parts of leech, 90 parts of Rhizoma Chuanxiong, 90 parts of Radix Angelicae Sinensis, 90 parts of safflower, 113 parts of peach kernel, 90 parts of radix paeoniae rubra, 90 parts of radix aucklandiae, stone
90 parts of calamus, 60 parts of pheretima, 90 parts of herba taxilli, 35 parts of Extractum Acanthopanacis Senticosi be prepared.
The detection method of content of effective component in a kind of HPLC-QQQ/MS method measurement Dragon groundnut provided by the invention,
The detection method of content includes the following steps:
(1), the preparation of test solution: taking Dragon groundnut content, and 60~80% methanol, ultrasound is added in finely ground mixing
20~40min is handled, with the weight of methanol supplement loss after letting cool, shakes up and takes supernatant, filter, used time dilution metering, and essence
It is close to weigh Indomethacin and Isorhamnetin reference substance, add 40~60% methanol solutions, dissolve and shake up, being configured to mass concentration is
The inner mark solution of 1~8 μ g/mL to get；
(2), the preparation of reference substance titer: precision weighs respectively, Paeoniflorin, ononin, eleutheroside E, hydroxyl safflower
Safflor yellow A, Calycosin-7-O-BETA-D-glucoside, Ligustilide, senkyunolide I, isofraxidin, amarogentin, oxypaeoniflorin, benzene first
Acyl Paeoniflorin, Astragaloside IV, ferulic acid, Syringin, Senkynolide H, dehydro-α-curcumene, calycosin, normal-butyl
Benzene peptide, Senkyunolide A, ligustrazine reference substance is appropriate, and 40~60% methanol are added and are dissolved, and it is molten to prepare mixing reference substance
Liquid；
(3), chromatographic condition: chromatographic column is using octadecyl silane column as filler, using 0.1% formic acid-water as mobile phase
A, using 0.1% formic acid-acetonitrile as Mobile phase B, volume proportion is mobile phase A phase: Mobile phase B phase are as follows: 80~0%:20~
100%, carry out gradient elution；Each sample introduction pre-equilibrates 2~10min, and flow velocity is 0.1~0.8mL/min, 2~6 μ L of sample volume；
(4), Mass Spectrometry Conditions: electric spray ion source (AJS-ESI), using multiple-reaction monitoring pattern, dry temperature degree 340~
360 DEG C, dry gas stream 8~10L/min of speed, 42~48psi of spray nozzle voltage, 230~270 DEG C of sheath temperature degree, sheath gas 9~
13L/min, positive and negative ion distinguish scan pattern, under positive ion mode in be designated as Indomethacin, under negative ion mode in be designated as it is different
Rhamnetin is internal standard；
(5), chromatographic peak measures: test sample (1) and (1) obtained and mixed reference substance solution by above-mentioned steps, according to above-mentioned
The Mass Spectrometry Conditions of chromatographic condition and step (4) of step (3), measure test liquid solution, obtain Dragon groundnut effective component from
Subflow chromatogram to get.
Preferably, the step is (1) in sample solution preparation method, and the methanol concentration is 75%, ultrasonic treatment
30min。
As a further preference, the step is (2) in the preparation of reference substance titer, and precision weighs each to be measured right respectively
According to product, the Paeoniflorin diluted concentration is 0.004~3 μ g/mL, and ononin diluted concentration is 0.1~2 μ g/mL, wilsonii
Glycosides E, hydroxyl radical carthamin yellow carthamus A, Calycosin-7-O-BETA-D-glucoside, Ligustilide, senkyunolide I, isofraxidin, amarogentin dilution
Concentration is 1 μ g/mL, and oxypaeoniflorin, Astragaloside IV, ferulic acid, Syringin, Senkynolide H, is gone benzoylpaeoniflorin
Hydrogen constuslactone diluted concentration is 0.1~0.5 μ g/mL, calycosin, n-butylbenzene peptide, Senkyunolide A diluted concentration
For 0.01~0.5 μ g/mL, ligustrazine diluted concentration is 0.01~0.05 μ g/mL, and is prepared into reference substance mixed solution.
Preferably, in the (3) chromatographic condition, the model Agilent Rapid Resolution HD of chromatographic column.
Preferably, the (3) chromatographic condition are as follows: 45 DEG C of used column temperature, mobile phase are A (0.1% formic acid-water)-B
(0.1% formic acid-acetonitrile), positive ion mode gradient elution 0-1.5min, 20%B；1.5-3min, 20%-80%B；3-
4min, 80%B；4-6min, 80%-100%B, negative ion mode gradient elution 0-0.5min, 20%B；0.5-1min,
20%-100%B；1-3 min, 100%B, each sample introduction pre-equilibrate 5min, flow velocity 0.5mL/min, 2 μ L of sample volume.
Preferably, the (4) Mass Spectrometry Conditions are as follows: electric spray ion source, using multiple-reaction monitoring pattern, dry temperature degree 350
DEG C, dry gas stream speed 9L/min, spray nozzle voltage 45psi, 250 DEG C, sheath gas 11L/min of sheath temperature degree, positive and negative ion is divided
Other scan pattern, is measured.
Chromatography of the present invention and Mass Spectrometry Conditions optimization
(1), mobile phase selects:
1., chromatographic column optimization: selection suitable chromatography column can make in herbal mixture Dragon groundnut mesh in active principle ingredient
Mark produce product preferably separate, and reach quick appearance, realize the quick analysis to sample compound.Investigate C18 chromatographic column
(3mm × 100 mm, 3 μm) and C18 chromatographic column (50mm × 2.1mm, 1.8 μm) two different chromatographic columns, select C18 chromatographic column
(50mm × 2.1 mm, 1.8 μm), can be realized the quick detection to sample；
2., adjustment chromatographic condition in eluent gradient ratio, on the one hand make analyze speed it is most short, on the other hand make with point
Isomers n butylphthalide, Ligustilide and senkyunolide I, H reach baseline separation.In order to by more Dragon groundnuts
In active principle ingredient detected be kept completely separate, we study in experiment is groped by a large amount of preliminary experiment, eventually
It is A (0.1% formic acid-water)-B (0.1% formic acid-acetonitrile) in find suitable optimal proportion of mobile phase be mobile phase, we
Some preferred chromatographic condition parameters are screened, to prove the creativeness of the present invention program.Such as: chromatographic condition 1:0-5min,
5%B；5-7min, 100%B (referring to attached drawing 3)；Chromatographic condition 2:0-1.5min, 20%B；1.5-1.6min, 20%-80%
B；1.6-5.3min, 80%-100%B；5.3-7min, 100%B (Fig. 4)；Chromatographic condition 3:0-1.5min, 20%B；1.5-
3min, 20%-80% B；3-4min, 80%B；4-6min, 80%-100%B (Fig. 5)；Grope by test of many times, it is last true
Fixation spectral condition 3 can be realized senkyunolide I and the baseline separation of H, while analysis time is shorter, it is thus determined that best stream
Dynamic phase gradient ratio is chromatographic condition 3.
(2), internal standard substance selects:
This experiment further increases the accuracy of measurement using internal standard method, close according to retention time, can in mass spectrum
Be ionized in fragmentation, sample there is no etc. principles carry out in target selection, investigated Indomethacin, carbamazepine, the network cheek
The compounds such as dimension, Isorhamnetin have finally determined that internal standard compound Isorhamnetin is Indomethacin, anion mould under positive ion mode
Internal standard compound is Isorhamnetin under formula.
(3), Mass Spectrometry Conditions optimization (MRM mode):
It needs to be determined that each compound parent ion, daughter ion, fragmentor value, CE value, steps are as follows for concrete analysis:
1., to each compound carry out mass spectrum Scan full scan, selection under full scan mode mass spectrum response it is stronger plus
It closes ion ([M+H]+、[M+Na]+、[M-H]-、[M+COOH]-) parent ion quantitative as respective compound, while determinization
It closes object best ion mode (positive and negative).
2., to each compound use mass spectrum SIM mode, it is known that different fragmentor values are set in the case of parent ion
(60-225V) selects the mass spectrum response under SIM mode stronger as the best fragmentor value of respective compound.
3., to each compound use mass spectrum product mode, it is known that parent ion and fragmentor value pass through setting
Different CE values (0-40V), select under product mode mass spectrum respond stronger fragmentation of ions as the compound son from
Son, while the mass spectrum of daughter ion responds the stronger conduct best CE value of respective compound in more different CE values.
4., to each compound use MRM mode, it is known that each compound parent ion, daughter ion, best fragmentor
Value and CE value obtain the mass spectra peak of the optimal compound.
Such as: by taking calycosin as an example:
1), mass spectrum Scan full scan determines calycosin [M+H] in the positive-ion mode+Respond stronger, selection [M+
H]+285 parent ion (referring to Fig. 6) as the compound,
2), using mass spectrum SIM mode, it is known that different fragmentor values is arranged in the case of parent ion 285, as a result hair stamen
Mass spectrum response is stronger when isoflavones fragmentor value is 165, determines that best fragmentor value is 165 (Fig. 7)；
3), using mass spectrum product mode, it is known that in the case of parent ion 285.1, fragmentor value 165, setting is different
Daughter ion for the compound is relatively pretended in CE value, as a result 213.2 mass spectrum of fragment ion response, under 40V daughter ion mass spectrum response compared with
By force, determine that best CE value is 40V (Fig. 8)；
4), using MRM mode, final to determine that calycosin ion pair is 285.1-213.2, fragmentor value is
165V, CE value are 40V (Fig. 9)；
Remaining compound mass spectrum Optimization Steps is same as above, final to determine Astragaloside IV, hydroxyl safflower yellow in Dragon groundnut
Plain A, calycosin glucose glucosides, calycosin, ferulic acid, Syringin, n butylphthalide, Senkyunolide A, ocean
Ligustilide I, Senkynolide H, ligustrazine, dehydro-α-curcumene, amarogentin, Indomethacin, Paeoniflorin, oxidation Chinese herbaceous peony
Glycosides, benzoylpaeoniflorin, Isorhamnetin, eleutheroside, isofraxidin, the specific attached drawing of Ligustilide compound mass spectrometry parameters are detailed
See Figure 10-Figure 30.
Compared with prior art, the invention has the following advantages:
(1), in current Dragon groundnut only using Astragaloside IV as the quality control index of Dragon groundnut, it is difficult to effectively
Characterize its active principle ingredient and inherent quality.And Chinese materia medica preparation pharmacological effect is the result of plurality of active ingredients synergistic effect.
It therefore should be the investigation of Multiple components for the performance rating of Dragon groundnut preparation, it is raw that the present invention establishes HPLC-QQQ/MS dragon
Leech capsule multi objective quality evaluation control method has the characteristics that quick, high sensitivity.This method can detect imperial life simultaneously
Astragaloside IV, hydroxyl radical carthamin yellow carthamus A, calycosin glucoside, calycosin, ferulic acid, lilac in leech capsule
Glycosides, n butylphthalide, Ligustilide, Senkyunolide A, senkyunolide I, Senkynolide H, ligustrazine, isofraxidin,
The Chinese medicine active ingredients such as dehydro-α-curcumene, amarogentin, eleutheroside E, Paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin
Ingredient.Showing detection method precision that the present invention establishes through test result, (withinday precision RSD range is in 0.43%-
4.91%, 19 kinds of determinand RSD ranges of day to day precision are in 3.12%-9.39%), (RSD range is in 1.52%- for stability
4.40%), repeatability (RSD range is in 0.28%-4.94%), the rate of recovery (rate of recovery 85%-114%, RSD value
0.61%-4.46%), it can be seen that, this method has operating method simple, high sensitivity, the accurate advantage of content.
(2), multicomponent quantitative analysis is to control the main method of compound preparation quality, this experiment reacts prison using mass spectrum more
Survey mode has primarily looked at ionization response of each compound in mass spectrum positive and negative ion mode, it is determined that Astragaloside IV etc.
Response is high in the positive-ion mode for 15 kinds of compounds, and 4 kinds of compound negative ion mode responses such as Paeoniflorin are high, therefore this
Experiment distinguishes method for measuring using positive and negative ion；Pass through mass spectrum Scan model selection parent ion, SIM model-based optimization capillary
Exit potential, Product model selection daughter ion optimize collision energy simultaneously, obtain each compound highest mass spectrum response, from
And determine each compound quota ion pair；In all compounds, senkyunolide I and Senkynolide H are different with point each other
(ion pair is isomer altogether each other for structure body (quota ion pair is 225-119.2 altogether), n butylphthalide and Ligustilide
191.3-145.1), so that this four compounds is reached baseline separation in order to which accurate quantitative analysis needs to optimize chromatographic condition, this test into
And adjust chromatographic isolation, the results showed that selection Agilent Rapid Resolution HD C18 (50mm × 2.1mm, 1.8 μ
M) the initial organic Phase Proportion of chromatographic column and adjustment can guarantee that this four compounds realize baseline separations, final foundation to 20%
To the quick quantitative analytic method of 19 kinds of ingredients of Dragon groundnut preparation in 9min.
(3), the present invention may be implemented using effective component in HPLC-QQQ/MS method measurement Dragon groundnut to the raw leech glue of dragon
The content Simultaneous Determination of 19 index components in capsule.The selected index components of the present invention, ferulic acid, Sydroxy carthamin
A, n butylphthalide, the effective components such as ligustrazine, modern pharmacology experiment show that it has expansion blood vessel and thrombus dissolving effect and dragon
The effect of raw leech, activating microcirculation and removing stasis medicinal, dredging collateral effect match, can objective characterisation its inherent quality so that Chinese patent drug material pesticide effect
Basis is definitely.In conclusion the raw leech HPLC-QQQ/MS detection method of content of dragon established by the present invention, have it is easy,
Accurately, efficiently, reproducibility, the characteristics of having good stability.The detection method of content can be controlled effectively in Dragon groundnut comprehensively
Reference is provided in quality.
Detailed description of the invention
Fig. 1-Dragon groundnut 19 kinds of active principle ingredients to be measured and internal standard compound multiple-reaction monitoring ion flow graph (cation mould
Formula)；
Fig. 2-Dragon groundnut 19 kinds of active principle ingredients to be measured and internal standard compound multiple-reaction monitoring ion flow graph (anion mould
Formula)；Wherein, chromatographic peak 1- Astragaloside IV；Chromatographic peak 2- hydroxyl radical carthamin yellow carthamus A；Chromatographic peak 3- calycosin glucoside；
Chromatographic peak 4- calycosin；Chromatographic peak 5- ferulic acid；Chromatographic peak 6- Syringin；Chromatographic peak 7- n butylphthalide；Chromatographic peak
8- Ligustilide；Chromatographic peak 9- Senkyunolide A；Chromatographic peak 10- senkyunolide I；Chromatographic peak 11- Senkynolide H；Color
Spectral peak 12- ligustrazine；Chromatographic peak 13- isofraxidin；Chromatographic peak 14- dehydro-α-curcumene；Chromatographic peak 15- amarogentin；Chromatography
Peak 16- eleutheroside E；Chromatographic peak 17- Paeoniflorin；Chromatographic peak 18- oxypaeoniflorin；Chromatographic peak 19- benzoylpaeoniflorin；Color
Spectral peak IS1Indomethacin；Chromatographic peak IS2- Isorhamnetin；
The MRM mass spectrogram of Fig. 3-senkyunolide I and Senkynolide H；
The MRM mass spectrogram of Fig. 4-senkyunolide I and Senkynolide H；
The MRM mass spectrogram of Fig. 5-senkyunolide I and Senkynolide H；
Fig. 6-calycosin Scan mode mass spectrogram；
Fig. 7-calycosin SIM mode mass spectrogram；
Fig. 8-calycosin Product mode mass spectrogram；
Fig. 9-calycosin MRM mass spectrogram；
Figure 10-Astragaloside IV mass spectrogram；
Figure 11-hydroxyl radical carthamin yellow carthamus A mass spectrogram；
Figure 12-calycosin glucose glucosides mass spectrogram；
Figure 13-calycosin mass spectrogram；
Figure 14-ferulic acid mass spectrogram；
Figure 15-Syringin mass spectrogram；
Figure 16-n butylphthalide mass spectrogram；
Figure 17-Senkyunolide A mass spectrogram；
Figure 18-senkyunolide I mass spectrogram；
Figure 19-Senkynolide H mass spectrogram；
Figure 20-ligustrazine mass spectrogram；
Figure 21-dehydro-α-curcumene mass spectrogram；
Figure 22-amarogentin mass spectrogram；
Figure 23-Indomethacin mass spectrogram；
Figure 24-Paeoniflorin mass spectrogram；
Figure 25-oxypaeoniflorin mass spectrogram；
Figure 26-benzoylpaeoniflorin mass spectrogram；
Figure 27-Isorhamnetin mass spectrogram；
Figure 28-eleutheroside E mass spectrogram；
Figure 29-isofraxidin mass spectrogram；
Figure 30-Ligustilide mass spectrogram；
19 kinds of chemical composition content distributions in 3 batches of Figure 31-Dragon groundnut.
Specific embodiment
In order to be more fully understood from implementation of the invention, the present invention is done further below by typical embodiment
It is bright.Unless otherwise defined, technical term or section used in present patent application specification and claims are academic
Language should be the ordinary meaning that persons with general skills in the field are understood.
Embodiment 1: the content detection side of effective component in the established HPLC-QQQ/MS method measurement Dragon groundnut of the present invention
1 instrument of method and reagent
1.1 instrument
Agilent1260 high performance liquid chromatograph, the triple quadrupole tandem mass spectrometer (U.S. Agilent6470
Agilent)；Assay balance (Mei Tele company of Switzerland)；3-18KS centrifuge (German Sigma)；KQ-500E type ultrasonic wave is clear
Washing machine (Kunshan Ultrasonic Instruments Co., Ltd.).
1.2 reagent
Reference substance Astragaloside IV (H-013-170614), hydroxyl radical carthamin yellow carthamus A (Q-008-170303), calycosin
Glucoside (M-020-170929), calycosin (M-021-170517), amarogentin (K-001-161216), asafoetide
Acid (A-002-161216), dehydro-α-curcumene (Q-016-170816), Syringin (C-009-161217), eleutheroside
E (C-008-171229), amarogentin (K-001-161216), Paeoniflorin (S-010-170214), oxypaeoniflorin (Q-
019-180315), benzoylpaeoniflorin (B-024-171216), ligustrazine (C-045-171216), Ligustilide (G-010-
171216), n-butylbenzene peptide (D-057-180321), Senkyunolide A (Y-083-170411), senkyunolide I (Y-
085-161010), Senkynolide H (Y-084-161013), isofraxidin (Y-027-170328), Isorhamnetin (internal standard,
Y-039-180420 Chinese Chengdu Rui Fensi Biotechnology Co., Ltd, Indomethacin (internal standard, Y18J7C18042)) are purchased from
It is purchased from source leaf biology, purity is all larger than 98%；Formic acid, acetonitrile and methanol are chromatographically pure (U.S. Fisher Chemical)；
Ultrapure water (Millipore company of the U.S.)；Dragon groundnut provides (lot number: 171250, by Shaanxi Buchang Pharmaceuticals Co., Ltd.
171251、171252)。
1.3 methods and result
1.3.1 the preparation of reference substance solution
Each reference substance to be measured is weighed, respectively plus the dissolution of 50% methanol is configured to 1mg/mL reference substance stock solution.Continue to add
Stock solution Paeoniflorin diluted concentration is 3 μ g/mL by 50% methanol, and ononin diluted concentration is 2 μ g/mL, eleutheroside E, hydroxyl
Base carthamus tinctorius yellow colour A, Calycosin-7-O-BETA-D-glucoside, Ligustilide, senkyunolide I, isofraxidin, amarogentin diluted concentration are 1
μ g/mL, oxypaeoniflorin, benzoylpaeoniflorin, Astragaloside IV, ferulic acid, Syringin, Senkynolide H, dehydrogenation radix aucklandiae
Lactone diluted concentration is 0.5 μ g/mL, and calycosin, n-butylbenzene peptide, Senkyunolide A diluted concentration are 0.1 μ g/mL,
Ligustrazine diluted concentration is 0.05 μ g/mL, prepares reference substance mixed solution.
1.3.2 the preparation of sample solution
The finely ground mixing of Dragon groundnut content is taken, powder 300mg is taken, 75% methanol of 30mL, ultrasonic treatment is added
30min shakes up with the weight of 75% methanol supplement loss after letting cool and takes supernatant, filtered with 0.22 μm of miillpore filter, the used time
Dilute 10 times of measurements.Precision weighs Indomethacin and Isorhamnetin reference substance is appropriate, adds 50% methanol solution, dissolves and shakes up,
It is configured to the inner mark solution that mass concentration is 4 μ g/mL.
1.3.3 chromatographic condition
Using Agilent Rapid Resolution HD C18 chromatographic column (50mm × 2.1mm, 1.8 μm), column temperature 45
DEG C, mobile phase is A (0.1% formic acid-water)-B (0.1% formic acid-acetonitrile), positive ion mode gradient elution 0-1.5min, 20%
B；1.5-3min, 20%-80%B；3-4min, 80%B；4-6min, 80%-100%B, negative ion mode gradient elution 0-
0.5min, 20% B；0.5-1min, 20%-100%B；1-3min, 100%B, each sample introduction pre-equilibrate 5min, and flow velocity is
0.5mL/min, 2 μ L of sample volume.
1.3.4 Mass Spectrometry Conditions
Electric spray ion source (AJS-ESI), using multiple-reaction monitoring pattern, dry 350 DEG C of temperature degree, dry gas stream speed
9L/min, spray nozzle voltage 45psi, 250 DEG C, sheath gas 11L/min of sheath temperature degree, positive and negative ion distinguishes scan pattern, just
Be designated as Indomethacin under ion mode, under negative ion mode in be designated as Isorhamnetin, untested compound the MS detection parameters are shown in
Table 1, untested compound ion stream chromatogram are shown in Fig. 1.
1 Dragon groundnut untested compound of table and internal standard compound the MS detection parameters
1.4 methodological study
1.4.1 linear relationship and quantitative limit
According to the reference substance stock solution operating method under " 1.3.1 " item, with 50% methanol according to 1:2:2:2:5:2:5:2:
5 dilution proportions are configured to graded series concentrations control product mixed solution, and inner mark solution is added before measuring.According to " 1.3.3 " item
Mass Spectrometry Conditions are measured under lower chromatographic condition and " 1.3.4 " item.Using reference substance mass concentration as abscissa (X), reference substance with
Internal standard peak area ratio is that ordinate (Y) carries out linear regression, obtains regression equation, related coefficient and the range of linearity, calculates simultaneously
The quantitative limit (S/N=10) (table 2) of each determinand.
2 Dragon groundnut untested compound linear relationship of table is investigated
1.4.2 precision
Mixed reference substance solution is taken, continuous sample introduction 6 times and measures in 1 day, is divided by above-mentioned testing conditions for three days on end
Analysis, record determinand and internal standard peak area, according to regression equation calculation concentration and RSD value, the results showed that withinday precision RSD
Range shows instrument precision in 3.12%-9.39% in 0.43%-4.91%, 19 kinds of determinand RSD ranges of day to day precision
Degree is good.
1.4.3 stability
Dragon groundnut sample solution is taken, respectively at 0,2,4,8,12, is analyzed for 24 hours by above-mentioned testing conditions, is recorded
Determinand and internal standard peak area, according to regression equation calculation concentration and RSD value, the results showed that 19 kinds of determinand RSD ranges exist
1.52%-4.40% shows that sample is stablized interior for 24 hours.
1.4.4 repeated
Same batch Dragon groundnut powder is taken, parallel 6 parts of preparation is analyzed by above-mentioned testing conditions, recorded to be measured
Object and internal standard peak area, according to regression equation calculation concentration and RSD value, the results showed that 19 kinds of determinand RSD ranges exist
0.28%-4.94% shows that the repeatability of this method is good.
1.4.5 the rate of recovery
Take the Dragon groundnut powder of known content 6 parts parallel, be separately added into equivalent reference substance, by above-mentioned testing conditions into
Row analysis, record determinand and internal standard peak area, according to regression equation calculation concentration and RSD value, the results showed that 19 kinds of determinands
Rate of recovery 85%-114%, RSD value 0.61%-4.46%, shows that the rate of recovery of method is good.
1.4.6 Dragon groundnut sample size measures
Graded series concentrations control product mixed solution and Dragon groundnut sample solution are taken, inner mark solution is added before measuring,
Assay is carried out simultaneously by above-mentioned testing conditions, the data of acquisition is imported into Agilent quantitative analysis software, dragon is calculated
The content (table 3) of 19 compounds in raw leech capsule specifies the content distribution (referring to attached drawing 31) of compound in the formulation, together
When calculate three batches of preparations in the RSD value of compound total content and the RSD value of each compounds content, specify compound difference batch
Stability existing for secondary preparation, and then the quality of the pharmaceutical preparations is evaluated.
19 chemical composition contents in 3 Dragon groundnut of table
19 kinds of compounds content ranges of Dragon groundnut of this measuring are 0.2-4125 μ g/g, wherein with Paeoniflorin and
Calycosin glucose Glycosides Contents highest, secondly be amarogentin, hydroxyl radical carthamin yellow carthamus A and Syringin, this 5 kinds at
Point total content up to 0.6% or more, cover monarch drug in a prescription Radix Astragali, ministerial drug radix paeoniae rubra, peach kernel, safflower and adjutant wilsonii it is main at
Point；Each compound total content of measurement is 5.19% in 3 batch preparation RSD values, while the content RSD of each compound
Value shows that the content of compound between different batches preparation is closer to, it can be seen that Dragon groundnut preparation matter less than 15%
Measure that relatively stable, technique is more mature；In addition, in all compounds, amarogentin, Paeoniflorin, oxypaeoniflorin, benzene first
Acyl Paeoniflorin and Syringin RSD value it is relatively small (RSD value is followed successively by 3.37%, 5.84%, 5.59%, 7.58%,
7.62%), show the relatively stable presence in different batches Dragon groundnut of these compounds, compound can in preparation
Stable deposit is the cardinal principle for formulating Chinese medicine quality control index.
Finally, it should be noted that the invention is not limited to above-mentioned specific embodiment, above-mentioned specific embodiment
It is only schematical, directiveness, rather than it is restrictive.Enlightenment of the those skilled in the art in this specification
Under, as long as within the scope of spirit and substance of the present invention, made any change, equivalent replacement and improvement, of the invention
Within protection scope.
Claims (6)
1. the detection method of content of effective component in a kind of HPLC-QQQ/MS method measurement Dragon groundnut, which is characterized in that described
Detection method of content includes the following steps:
(1), the preparation of test solution: taking Dragon groundnut content, and 60~80% methanol, ultrasonic treatment is added in finely ground mixing
20~40min shakes up with the weight of methanol supplement loss after letting cool and takes supernatant, filter, used time dilution metering, and accurate title
Indomethacin and Isorhamnetin reference substance are taken, 40~60% methanol solutions are added, dissolves and shakes up, being configured to mass concentration is 1~8
The inner mark solution of μ g/mL to get；
(2), the preparation of reference substance titer: precision weighs respectively, Paeoniflorin, ononin, eleutheroside E, hydroxyl safflower yellow
Plain A, Calycosin-7-O-BETA-D-glucoside, Ligustilide, senkyunolide I, isofraxidin, amarogentin, oxypaeoniflorin, benzoyl Chinese herbaceous peony
Glycosides, Astragaloside IV, ferulic acid, Syringin, Senkynolide H, dehydro-α-curcumene, calycosin, n-butylbenzene peptide, ocean
4,5-dihydro-3-butylidene-phthalide, ligustrazine reference substance is appropriate, and 40~60% methanol are added and are dissolved, mixed reference substance solution is prepared；
(3), chromatographic condition: chromatographic column is using octadecyl silane column as filler, using 0.1% formic acid-water as mobile phase A, with
0.1% formic acid-acetonitrile is Mobile phase B, and volume proportion is mobile phase A phase: Mobile phase B phase are as follows: 80~0%:20~100%,
Carry out gradient elution；Each sample introduction pre-equilibrates 2~10min, and flow velocity is 0.1~0.8mL/min, 2~6 μ L of sample volume；
(4), Mass Spectrometry Conditions: electric spray ion source (AJS-ESI), using multiple-reaction monitoring pattern, dry temperature degree 340~360
DEG C, dry gas stream 8~10L/min of speed, 42~48psi of spray nozzle voltage, 230~270 DEG C of sheath temperature degree, 9~13L/ of sheath gas
Min, positive and negative ion distinguish scan pattern, under positive ion mode in be designated as Indomethacin, under negative ion mode in be designated as different sandlwood
Element is internal standard；
(5), chromatographic peak measures: test sample (1) and (1) obtained and mixed reference substance solution by above-mentioned steps, according to above-mentioned steps
(3) the Mass Spectrometry Conditions of chromatographic condition and step (4) measure test liquid solution, obtain the ion stream of Dragon groundnut effective component
Chromatogram to get.
2. detection method of content according to claim 1, which is characterized in that the step (1) sample solution preparation method
In, the methanol concentration is 75%, is ultrasonically treated 30min.
3. detection method of content according to claim 1, which is characterized in that the preparation of the step (2) reference substance titer
In, precision weighs each reference substance to be measured respectively, and the Paeoniflorin diluted concentration is 0.004~3 μ g/mL, and ononin dilution is dense
Degree be 0.1~2 μ g/mL, eleutheroside E, hydroxyl radical carthamin yellow carthamus A, Calycosin-7-O-BETA-D-glucoside, Ligustilide, senkyunolide I,
Isofraxidin, amarogentin diluted concentration are 1 μ g/mL, oxypaeoniflorin, benzoylpaeoniflorin, Astragaloside IV, ferulic acid, purple
Syringin, Senkynolide H, dehydro-α-curcumene diluted concentration be 0.1~0.5 μ g/mL, calycosin, n-butylbenzene peptide,
Senkyunolide A diluted concentration is 0.01~0.5 μ g/mL, and ligustrazine diluted concentration is 0.01~0.05 μ g/mL, and is prepared into
Reference substance mixed solution.
4. detection method of content according to claim 1, which is characterized in that in the (3) chromatographic condition, the model of chromatographic column
For Agilent Rapid Resolution HD.
5. detection method of content according to claim 1, which is characterized in that the (3) chromatographic condition are as follows: used column temperature
45 DEG C, mobile phase is A (0.1% formic acid-water)-B (0.1% formic acid-acetonitrile), positive ion mode gradient elution 0-1.5min,
20%B；1.5-3min, 20%-80%B；3-4min, 80%B；4-6min, 80%-100%B, negative ion mode gradient elution
0-0.5min, 20%B；0.5-1min, 20%-100%B；1-3min, 100%B, each sample introduction pre-equilibrate 5min, and flow velocity is
0.5mL/min, 2 μ L of sample volume.
6. detection method of content according to claim 1, which is characterized in that the (4) Mass Spectrometry Conditions are as follows: electric spray ion source,
Using multiple-reaction monitoring pattern, dry 350 DEG C of temperature degree, dry gas stream speed 9L/min, spray nozzle voltage 45psi, sheath temperature degree 250
DEG C, sheath gas 11L/min, positive and negative ion distinguishes scan pattern, is measured.
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