CN104614456B - A kind of method simultaneously detecting blood plasma midbrain Xintong capsule main component - Google Patents
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Abstract
The invention provides a kind of liquid chromatography tandem mass spectrometry (HPLC MS/MS) and detect plasma sample midbrain Xintong capsule main component Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, the method for tanshinone IIA simultaneously.In described liquid chromatogram, flowing is made up of acetonitrile and the aqueous formic acid that volume fraction is 0.1%, uses gradient elution.Described mass spectrum uses negative ions to be switched fast analytical model, MRM scan mode.Measure after being administered cerebral ischemic capsule, the blood concentration situation of change of these several main components in rat plasma simultaneously.Methodological study result shows that set up method meets vivo biodistribution sample determination requirement, and method sensitivity is good, and specificity is strong, stable, reliable, the detection of the relatively low material of Optimum Contents.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of method simultaneously detecting blood plasma midbrain Xintong capsule main component
And the application in pharmacokinetics.
Background technology
Cerebral ischemic capsule is to be produced without competition by Shaanxi Buchang Pharmaceuticals Co., Ltd., and its national drug authentication code is that traditional Chinese medicines are accurate
Word Z20090527.Cerebral ischemic capsule is by the Radix Astragali, the radix paeoniae rubrathe, the red sage root, Radix Angelicae Sinensis, Ligusticum wallichii, peach kernel, safflower, frankincense (system), myrrh
(system), reticulate millettia, the root of bidentate achyranthes, cassia twig, ramulus mori, earthworm, scorpio, leech, the listing Chinese patent drug of ten Six-element Chinese medicine compositions.Clinical main
For apoplexy apoplex involving the channels and collaterals caused by blood stagnancy due to deficiency of QI, venation block, the strong language of hemiplegia, extremity numbness, facial paralysis, tongue is stuttering and the obstruction of qi in the chest
Pained, uncomfortable in chest, palpitaition, breathe hard；Cerebral infarction, coronary disease and angina pectoris belong to above-mentioned patient.Document reports that it is mainly composed of the Radix Astragali
Saponins, phenolic acid class, tanshinone, flavonoids etc., and astragaloside, phenolic acid class, tanshinone component containing in tablets
Measure higher.Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, awns handle
Xanthin, Cryptotanshinone, tanshinone IIA, be in the Radix Astragali, the radix paeoniae rubrathe, the red sage root, Radix Angelicae Sinensis, Ligusticum wallichii, peach kernel, reticulate millettia, ramulus mori respectively
Main component, its pharmacologically active is reported by lot of documents.Simultaneously dynamic to the medicine generation of these several compositions but without document
Mechanics is reported, the article of cerebral ischemic capsule effective substance basic research also be there are no report.
Summary of the invention
It is an object of the invention to provide a kind of liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and detect plasma sample simultaneously
The main component of midbrain Xintong capsule, such as Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, red phenol
The method of the compositions such as acid B, Astragaloside IV, formononetin, Cryptotanshinone, tanshinone IIA.And use the method to the aforementioned brain heart
Main component in open capsule carries out pharmacokinetic, to further clear and definite cerebral ischemic capsule components ingested into the blood and
Its blood concentration situation of change, thus provide safeguard for the drug safety of cerebral ischemic capsule, medication effect.
Method provided by the present invention includes: the preparation of step (1) sample and step (2) use Liquid Chromatography-Tandem Mass Spectrometry
Method detects；
The method that the preparation employing of step (1) sample comprises the following steps:
A) in test plasma sample, add mixing inner mark solution, formic acid, and mix；
B) add acetonitrile precipitation albumen, stand after mixing；
C) centrifugal, collect supernatant, nitrogen dries up, and collects dried object；
D) the dried object methyl alcohol obtained is redissolved, mix, be centrifuged, take supernatant.
In step a), described in described mixing inner mark solution in be designated as chlorogenic acid, Gardenoside, Sennoside B, Puerarin.Institute
State mixing inner mark solution especially by the method preparation comprised the steps: precision weighs chlorogenic acid, Gardenoside, Sennoside B, Pueraria lobota
The root each 1mg of element, in 10mL volumetric flask, adds proper amount of methanol and dissolves, and be settled to graduation mark, be made into internal standard chlorogenic acid, Cape jasmine respectively
Sub-glycosides, Sennoside B, the storing solution of Puerarin；It is appropriate that precision pipettes each internal standard storing solution, with methanol dilution, is configured to containing green former
Acid, Gardenoside, Sennoside B, Puerarin are the mixing inner mark solution of 1.00 μ g/mL-1.
In described step a), described test plasma sample is 100:10 with the volume ratio of formic acid.
In described step b), the time of described standing is 2-5min.Described test plasma sample with the volume ratio of acetonitrile is
1:4。
In described step d), may also include before centrifugation step and the product after mixing is carried out ultrasonic step.
The described ultrasonic time is 30-60s.
Step a), b), c) and d) in, described mixing all uses the mode of vortex, and the time of vortex is 1-2min.
Step c) and d) in, described centrifugal condition is 18000 × g-19000 × g and is centrifuged 10-15min.
Step (2) detection liquid phase chromatogram condition used is as follows:
Chromatographic column is C18 post；
Flowing is made up of the aqueous formic acid that acetonitrile and volume fraction are 0.1% (v/v)；
Type of elution is gradient elution；
The program of described gradient elution is as follows:
It is constant that the volume fraction of 0-10min: acetonitrile maintains 30%；
The volume fraction of 10-15min: acetonitrile is increased to 80% by 30%；
The volume fraction of 15-20min: acetonitrile is increased to 95% by 80%；
It is constant that the volume fraction of 20-30min: acetonitrile maintains 95%；
Step (2) detection Mass Spectrometry Conditions used is as follows: use API 3200TMLC/MS/MS liquid matter analyzes system；
Ion gun, negative ions is switched fast analytical model, MRM scan mode.
In described liquid phase chromatogram condition, chromatographic column be specially Agilent Eclipse Plus C18 post (4.6mm ×
100mm,1.8μm)；Flow rate of mobile phase is 300 μ L/min；Column temperature is 30 DEG C；Sample size is 10 μ L.
In described Mass Spectrometry Conditions, described negative ions is switched fast in analytical model, and positive ion mode parameter is set to: gas
Curtain gas (CUR): 10psi；Collision gas (CAD): 80psi；Ion spray voltage (IS): 5000V；Temperature (TEM): 500 DEG C；Air-flow
1 (GS1): 40psi；Air-flow 2 (GS2): 30psi；Heating (Ihe): On；Negative ion mode parameter is set to: gas curtain gas (CUR):
20psi；Collision gas (CAD): 8psi；Ion spray voltage (IS) :-4500V；Temperature (TEM): 500 DEG C；Air-flow 1 (GS1):
30psi；Air-flow 2 (GS2): 30psi；Heating (Ihe): On.
The parameter of described MRM method is as follows:
Paeoniflorin: parent ion mass number (Q1): 478.6；Fragment ion masses number (Q3): 121.1；Solve bunch voltage (DP) :-
45V；Entrance potential (EP) :-4V；Sampling time (Dwell Time): 100；Impact energy :-30V；Collision cell exit potential
(CXP) :-1V；Retention time (Rt): 4.47min；
β ecdysterone: parent ion mass number (Q1): 478.9；Fragment ion masses number (Q3): 159.0；Solve bunch voltage
(DP) :-60V；Entrance potential (EP) :-10V；Sampling time (Dwell Time): 100；Impact energy :-37V；Collision cell outlet electricity
Pressure (CXP) :-1V；Retention time (Rt): 4.48min；
Amarogentin: parent ion mass number (Q1): 455.8；Fragment ion masses number (Q3): 322.8；Solve bunch voltage :-
50V；Entrance potential (EP) :-4.5V；Sampling time (Dwell Time): 100；Impact energy :-20V；Collision cell exit potential
(CXP) :-22V；Retention time (Rt): 4.49min；
Mulberroside A: parent ion mass number (Q1): 567.0；Fragment ion masses number (Q3): 242.6；Solve bunch voltage: 60V；
Entrance potential (EP): 10V；Sampling time (Dwell Time): 100；Impact energy: 38V；Collision cell exit potential (CXP): 16V；
Retention time (Rt): 4.53min；
Caffeic acid: parent ion mass number (Q1): 178.9；Fragment ion masses number (Q3): 134.9；Solve bunch voltage: 35V；
Entrance potential (EP): 3.5V；Sampling time (Dwell Time): 100；Impact energy: 24V；Collision cell exit potential (CXP):
0.5V；Retention time (Rt): 5.28min；
Forulic acid: parent ion mass number (Q1): 192.8；Fragment ion masses number (Q3): 133.9；Solve bunch voltage :-31V；
Entrance potential (EP) :-4V；Sampling time (Dwell Time): 100；Impact energy :-23V；Collision cell exit potential (CXP) :-
1V；Retention time (Rt): 7.32min；
Tanshin polyphenolic acid B: parent ion mass number (Q1): 716.5；Fragment ion masses number (Q3): 518.5；Solve bunch voltage :-
55V；Entrance potential (EP) :-4.5V；Sampling time (Dwell Time): 100；Impact energy :-25V；Collision cell exit potential
(CXP) :-40V；Retention time (Rt): 8.92min.
Astragaloside IV: parent ion mass number (Q1): 807.0；Fragment ion masses number (Q3): 807.0；Solution bunch voltage:
140V；Entrance potential (EP): 10V；Sampling time (Dwell Time): 100；Impact energy: 50V；Collision cell exit potential
(CXP): 55V；Retention time (Rt): 18.79min.
Formononetin: parent ion mass number (Q1): 267.0；Fragment ion masses number (Q3): 251.8；Solve bunch voltage :-
16V；Entrance potential (EP) :-2V；Sampling time (Dwell Time): 100；Impact energy :-27V；Collision cell exit potential
(CXP) :-5V；Retention time (Rt): 20.43min.
Cryptotanshinone: parent ion mass number (Q1): 297.0；Fragment ion masses number (Q3): 254.2；Solution bunch voltage:
50V；Entrance potential (EP): 9V；Sampling time (Dwell Time): 100；Impact energy: 35V；Collision cell exit potential (CXP):
15V；Retention time (Rt): 25.64min.
Tanshinone IIA: parent ion mass number (Q1): 295.2；Fragment ion masses number (Q3): 277.1；Solution bunch voltage:
58V；Entrance potential (EP): 10V；Sampling time (Dwell Time): 100；Impact energy: 27V；Collision cell exit potential (CXP):
21V；Retention time (Rt): 27.53min.
Gardenoside (IS): parent ion mass number (Q1): 387.0；Fragment ion masses number (Q3): 224.7；Solve bunch voltage :-
40V；Entrance potential (EP) :-9V；Sampling time (Dwell Time): 100；Impact energy :-15V；Collision cell exit potential
(CXP) :-15V；Retention time (Rt): 4.47min.
Sennoside B (IS): parent ion mass number (Q1): 860.6；Fragment ion masses number (Q3): 385.9；Solve bunch electricity
Pressure :-87V；Entrance potential (EP) :-10V；Sampling time (Dwell Time): 100；Impact energy :-60V；Collision cell exit potential
(CXP) :-2V；Retention time (Rt): 4.43min.
Puerarin (IS): parent ion mass number (Q1): 414.8；Fragment ion masses number (Q3): 266.9；Solve bunch voltage :-
50V；Entrance potential (EP) :-9V；Sampling time (Dwell Time): 100；Impact energy :-46V；Collision cell exit potential
(CXP) :-2V；Retention time (Rt): 4.44min.
Chlorogenic acid (IS): parent ion mass number (Q1): 352.8；Fragment ion masses number (Q3): 190.9；Solve bunch voltage :-
33V；Entrance potential (EP) :-4V；Sampling time (Dwell Time): 100；Impact energy :-32V；Collision cell exit potential
(CXP) :-2V；Retention time (Rt): 4.45min.
Present invention additionally comprises a kind of liquid chromatography tandem mass spectrometry detect simultaneously Paeoniflorin in plasma sample, β ecdysterone,
Amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, tanshinone IIA
The method of content, specifically include following step:
1) preparation of calibration curve: take the Paeoniflorin of a series of concentration, β ecdysterone, amarogentin, Mulberroside A, coffee
Acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, the hybrid standard product solution of tanshinone IIA add
In blank plasma samples, it is prepared according to above-mentioned sample preparation methods, then to the supernatant obtained according to above-mentioned liquid
Phase chromatographic tandem mass spectrography detects, and records the Paeoniflorin of each concentration, β ecdysterone, amarogentin, mulberry skin glucoside respectively
The peak area that A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, tanshinone IIA are corresponding；With
Paeoniflorin and internal standard peak area ratio Y are ordinate, with Paeoniflorin concentration X as abscissa, prepare the linear regression side of Paeoniflorin
Journey；With β ecdysterone and internal standard peak area ratio Y as ordinate, with β ecdysterone concentration X as abscissa, preparation β is casted off a skin steroid
The equation of linear regression of ketone；With amarogentin and internal standard peak area ratio Y as ordinate, with amarogentin concentration X as abscissa,
Prepare the equation of linear regression of amarogentin；With Mulberroside A and internal standard peak area ratio Y as ordinate, with Mulberroside A concentration X
For abscissa, prepare the equation of linear regression of Mulberroside A；With caffeic acid and internal standard peak area ratio Y as ordinate, with caffeic acid
Concentration X is abscissa, prepares caffeinic equation of linear regression；With forulic acid and internal standard peak area ratio Y as ordinate, with Ah
Wei, acid concentration X was abscissa, prepared the equation of linear regression of forulic acid；Sit for vertical with tanshin polyphenolic acid B with internal standard peak area ratio Y
Mark, with tanshin polyphenolic acid B concentration X as abscissa, prepares the equation of linear regression of tanshin polyphenolic acid B；With Astragaloside IV and internal standard peak area ratio
Value Y is ordinate, with Astragaloside IV concentration X as abscissa, prepares the equation of linear regression of Astragaloside IV；With formononetin with
Internal standard peak area ratio Y is ordinate, with formononetin concentration X as abscissa, prepares the linear regression side of formononetin
Journey；With Cryptotanshinone and internal standard peak area ratio Y as ordinate, with Cryptotanshinone concentration X as abscissa, prepare Cryptotanshinone
Equation of linear regression；With tanshinone IIA and internal standard peak area ratio Y as ordinate, with tanshinone IIA concentration X as abscissa, system
The equation of linear regression of standby tanshinone IIA；
2) Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, red phenol in test plasma sample
Acid B, Astragaloside IV, formononetin, Cryptotanshinone, the assay of tanshinone IIA: by test plasma sample according to above-mentioned
Sample preparation methods is prepared, and then detects the supernatant obtained according to above-mentioned liquid chromatography tandem mass spectrometry,
And record Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, awns respectively
The peak area that handle xanthin, Cryptotanshinone, tanshinone IIA are corresponding；Calculate Paeoniflorin and internal standard peak area ratio Y, Y value is substituted into
In the equation of linear regression of described Paeoniflorin, it is calculated the concentration of Paeoniflorin in described test plasma sample；Calculate β to cast off a skin steroid
Ketone and internal standard peak area ratio Y, substitute into Y value in the equation of linear regression of described β ecdysterone, be calculated described blood to be measured
The concentration of β ecdysterone in slurry samples；Calculate amarogentin and internal standard peak area ratio Y, Y value is substituted into described amarogentin
In equation of linear regression, it is calculated the concentration of amarogentin in described test plasma sample；Calculate Mulberroside A and face, internal standard peak
Long-pending ratio Y, substitutes into Y value in the equation of linear regression of described Mulberroside A, is calculated mulberry skin glucoside in described test plasma sample
The concentration of A；Calculate caffeic acid and internal standard peak area ratio Y, Y value is substituted in described caffeinic equation of linear regression, calculate
Obtain caffeinic concentration in described test plasma sample；Calculate forulic acid and internal standard peak area ratio Y, Y value is substituted into described
In the equation of linear regression of forulic acid, it is calculated the concentration of forulic acid in described test plasma sample；Calculate tanshin polyphenolic acid B with interior
Mark peak area ratio Y, substitutes into Y value in the equation of linear regression of described tanshin polyphenolic acid B, is calculated in described test plasma sample
The concentration of tanshin polyphenolic acid B；Calculate Astragaloside IV and internal standard peak area ratio Y, Y value is substituted into the linear regression side of described Astragaloside IV
Cheng Zhong, is calculated the concentration of Astragaloside IV in described test plasma sample；Calculate formononetin and internal standard peak area ratio
Y, substitutes into Y value in the equation of linear regression of described formononetin, is calculated rest-harrow in described test plasma sample yellow
The concentration of element；Calculate Cryptotanshinone and internal standard peak area ratio Y, Y value substituted in the equation of linear regression of described Cryptotanshinone,
It is calculated the concentration of Cryptotanshinone in described test plasma sample；Calculate tanshinone IIA and internal standard peak area ratio Y, by Y value
Substitute in the equation of linear regression of described tanshinone IIA, be calculated the concentration of tanshinone IIA in described test plasma sample.
The method of the invention can be used successfully to cerebral ischemic capsule main component (Paeoniflorin, β ecdysterone, amarogentin,
Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, tanshinone IIA) medicine generation dynamic
Mechanics study.
Described cerebral ischemic capsule is made up of the raw material of following masses part: the Radix Astragali 66 weight portion, the radix paeoniae rubrathe 27 weight portion, the red sage root 27
Weight portion, Radix Angelicae Sinensis 27 weight portion, Ligusticum wallichii 27 weight portion, peach kernel 27 weight portion, safflower 13 weight portion, frankincense 13 weight portion, system
Myrrh 13 weight portion, reticulate millettia 20 weight portion, the root of bidentate achyranthes 27 weight portion, cassia twig 20 weight portion, ramulus mori 27 weight portion, earthworm 27 weight
Part, scorpio 13 weight portion, leech 27 weight portion composition.By earthworm, scorpio, it is ground into fine powder；Remaining Radix Astragali etc. 14 taste is pulverized
Become fine powder, with earthworm, scorpion facing-up, sieve, mixing, load capsule, obtain capsule.
The present invention establishes the rat plasma sample processing method of protein precipitation, uses LC-MS (LC-ESI-MS/MS)
Analytic approach, measure simultaneously and be administered after cerebral ischemic capsule, Paeoniflorin, β ecdysterone, amarogentin, mulberry skin glucoside in rat plasma
A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, the blood concentration change of tanshinone IIA
Situation, and it is carried out methodological study.Result shows that set up method meets vivo biodistribution sample determination requirement, method spirit
Sensitivity is good, and specificity is strong, stable, reliable, the detection of the relatively low material of Optimum Contents.
HPLC-MS/MS detection plasma sample midbrain Xintong capsule main component Paeoniflorin, β that the present invention is set up are casted off a skin
Sterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, the red sage root
The method of ketone II A, has that method sensitivity is good, specificity is strong, the reliable feature of stability, specifically has a following beneficial effect:
1, main 11 of the HPLC-MS/MS detection plasma sample midbrain Xintong capsule set up by the present invention enter blood
Active principle composition, and have selected Gardenoside, sennoside, Puerarin, chlorogenic acid as internal standard, measures Paeoniflorin, β casts off a skin steroid
Ketone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, tanshinone
The blood concentration-time curve of II A, determines the pharmacokinetic parameter of each composition.These experimental results disclose the most further
Cerebral ischemic can effectively treat the active principle components group of cardiovascular and cerebrovascular disease it was confirmed Chinese medicine compound prescription multicomponent Synergistic treatment disease
Material base；Wherein technical solution of the present invention select with forulic acid, tanshin polyphenolic acid B, Cryptotanshinone, tanshinone IIA for measure refer to
Mark composition, modern pharmacology experiment shows, these compositions can suppress platelet aggregation, and can be obviously improved cerebral ischemia reperfusion damage
Hindering the neurological deficit of rat, it has antithrombotic effect, and it is similar that this has function promoting blood circulation and removing blood stasis with cerebral ischemic, this
A little achievements, this is also the research of the pharmacokinetics being cerebral ischemic oral administration further, establishes solid foundation, has important
Directive significance.
2, investigated the specificity checking of plasma sample midbrain Xintong capsule main component, precision, the degree of accuracy, extraction are returned
Yield, matrix effect, minimum quantitative limit and stability, methodology result shows, measured 11 target-finding compounds and 4
The separating degree of individual internal standard composition is high, and peak shape is good, without endogenous material interference measurement in blood plasma；11 kinds of compounds minimum fixed
Amount limit is all from this explanation of 0.075-10ng mL-1, and the analysis method of foundation has higher sensitivity, can be used for low concentration in blood plasma
The detection of medicine；Measure Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Radix Astragali first
Glycosides, formononetin, Cryptotanshinone, the LC-MS/MS of tanshinone IIA analyze method and have the preferable degree of accuracy and precision, meet
Determination of Biological Samples precision requirement；Plasma sample completes sample determination within 2 week, and each compound results RSD value is equal
Meet the requirements, show that set up method has good stability.
3, the HPLC-MS/MS detection method using the present invention to be set up, measures administration rat hindbrain Xintong capsule and mainly becomes
The blood concentration-time curve divided, result confirms, after Oral Administration in Rats cerebral ischemic capsule, caffeic acid, forulic acid, rest-harrow Huang
Element, Cryptotanshinone, tanshinone IIA all can absorbed into serum, caffeic acid, forulic acid, formononetin have absorption fast and eliminate fast,
Cryptotanshinone, tanshinone IIA have absorption fast and eliminate the features such as slower；Simultaneously along with the increase of dosage, five kinds of compounds
AUC also becomes big, and showing that it absorbs increases.Also further illustrate cerebral ischemic active principle component substances metabolic process, set up
Method have that sensitivity is good, specificity is strong, stable, accurate, reliable, can the detection of the relatively low material of Optimum Contents.
4, the HPLC-MS/MS detection method that the present invention is set up can disposably detect the multiple work in cerebral ischemic capsule
Property composition plasma drug concentration data, and testing result is accurate, highly sensitive.
Accompanying drawing explanation
Fig. 1 is the chromatogram of the plasma sample measured, and wherein, (A) is blank plasma chromatogram；(B) it is in blank plasma
Add each compound and interior target chromatogram；(C) it is that rat is to the blood plasma chromatogram of 30min after 1.5g/kg cerebral ischemic open capsule.
Fig. 2 is caffeinic blood concentration-time curve in rat plasma.
Fig. 3 is the blood concentration-time curve of forulic acid in rat plasma.
Fig. 4 is the blood concentration-time curve of formononetin in rat plasma.
Fig. 5 is the blood concentration-time curve of Cryptotanshinone in rat plasma.
Fig. 6 is the blood concentration-time curve of tanshinone IIA in rat plasma.
Detailed description of the invention
Below by specific embodiment, the present invention will be described, but the invention is not limited in this.
Experimental technique used in following embodiment if no special instructions, is conventional method；Institute in following embodiment
Reagent, biomaterial etc., if no special instructions, the most commercially obtain.
Cerebral ischemic capsule (lot number is 20121017) used in following embodiment is carried by Shaanxi Buchang Pharmaceuticals Co., Ltd.
Supply.Reagent used in following embodiment:
Acetonitrile (U.S. Fisher),
Ultra-pure water (Millipore ultra-pure water cleaning system prepares),
Methyl alcohol (chromatogram rank, Tianjin Concord Technology Co., Ltd.),
Ethyl acetate (analyzes pure rank, Tianjin Concord Technology Co., Ltd.),
Formic acid (high-purity, content > 99.99%, Tianjin recovery fine chemistry industry research institute).
Instrument used in following embodiment:
LC-MS instrument: Agilent 1200 high performance liquid chromatograph, API 3200 concatenates quadrupole mass spectrometer；
Superpure water machine: Millipore company, Mill-Q II type；
Supercentrifuge: Sigma Co., USA, 3K15；
Ultra low temperature freezer: Thermo Heto company of the U.S. ,-86 DEG C；
Ultrasonic cleaner: Kunshan Ultrasonic Instruments Co., Ltd., KQ-250E；
Nitrogen evaporator: Organomation company of the U.S., N-EAVPTM111；
100000/balance: Mettler Toledo company of Switzerland, AX205；
Ten thousand/balance: Sartorius company of Germany, BP121S；
Vortex mixer: Shanghai Hu Xi analytical instrument factory, XW-80A.
Experimental animal used in following embodiment:
Healthy male rat, strain SD, rank SPF level, body weight 200 ± 20g, purchased from mountains and rivers, Tianjin red animal used as test section
Skill Co., Ltd.
Five kinds of determinand standard items used in following embodiment: Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A,
Caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, tanshinone IIA (China's pharmaceutical biological product
Calibrating institute, purity ＞ 98%).
Internal standard: Gardenoside, Sennoside B, Puerarin, chlorogenic acid (Nat'l Pharmaceutical & Biological Products Control Institute, purity ＞ 98%).
First the method for ethyl acetate liquid-liquid extraction is optimized by this research, but due to determinand polarity spectrum relatively
Greatly, so the part determinand rate of recovery and matrix effect can not meet the requirement of biological sample analysis.
In addition the method having investigated acetonitrile precipitation albumen, experiment adds formic acid on the basis of acetonitrile precipitation albumen, examines
Examined formic acid add volume (0,5,10 and 20) on experiment impact, result shows, the method for acetonitrile precipitation albumen compares acetic acid
The method of ethyl ester extraction is more preferable to the rate of recovery and the matrix effect of analysis ingredient, and when adding 10 μ L formic acid, sample and acetonitrile exist
Under the ratio of 1:4, sedimentation effect is preferable, and the addition of formic acid significantly improves the rate of recovery of analysis ingredient, and the degree of accuracy of method is relatively
Good.Therefore use acetonitrile precipitation albumen that plasma sample is processed, have that method is simple, low cost and other advantages.In sum, originally
Experimental selection adds 10 μ L formic acid, uses acetonitrile to carry out protein precipitation, before plasma sample is carried out by the method that 100% methyl alcohol redissolves
Process, it is achieved that to the isolation and purification measuring composition.
Embodiment 1, the foundation of plasma sample midbrain Xintong capsule Principle components analysis method
1.1 LC-MS/MS conditions:
Chromatographic column is Agilent Eclipse Plus C18 post (4.6mm × 100mm, 1.8 μm)；Guard column is
Agilent C18 post (2.1 × 12.5mm, 5 μm)；Flowing is mutually: A: acetonitrile, B: water (containing volume fraction 0.1% formic acid)；Flow velocity
It is 300 μ L/min；Column temperature is 30 DEG C；Sample size is 10 μ L；Time is 0-30min；
Using gradient elution, elution program is shown in Table 1.
Table 1 eluent gradient elution program
Mass Spectrometry Conditions: use API 3200TMLC/MS/MS liquid matter analyzes system；Ion gun, positive and negative
Ion is switched fast analytical model, MRM scan mode；
Wherein positive ion mode: gas curtain gas (CUR): 10psi；Collision gas (CAD): 8psi；Ion spray voltage (IS):
5000V；Temperature (TEM): 500 DEG C；Air-flow 1 (GS1): 40psi；Air-flow 2 (GS2): 30psi；Heating (Ihe): On；Anion mould
Formula: gas curtain gas (CUR): 20psi；Collision gas (CAD): 8psi；Ion spray voltage (IS) :-4500V；Temperature (TEM): 500
℃；Air-flow 1 (GS1): 30psi；Air-flow 2 (GS2): 30psi；Heating (Ihe): On.
Described MRM method parameter is shown in Table 2.
Table 2 compound mass spectrometry parameters
The preparation of 1.2 solution
1.2.1 the preparation of standard items storing solution: precision weighs Paeoniflorin 1.00mg, β ecdysterone 1.00mg, semen armeniacae amarae
Glycosides 1.00mg, Mulberroside A 1.00mg, caffeic acid 1.00mg, forulic acid 1.00mg, tanshin polyphenolic acid B 1.00mg, Astragaloside IV
1.00mg, formononetin 1.00mg, Cryptotanshinone 1.00mg, tanshinone IIA 1.00mg, in 10mL volumetric flask, add suitable
Amount methyl alcohol dissolves, and is settled to graduation mark, is made into each standard items storing solution；
1.2.2 the preparation of standard items mixed solution: each standard items storing solution of accurate shifting is appropriate, with methanol dilution, prepares dense
Degree is respectively Paeoniflorin 2.5-750ng/mL；β ecdysterone 5-1500ng/mL；Amarogentin 10-3000ng/mL；Mulberry skin glucoside
A3-3000ng/mL；Caffeic acid 3-3000ng/mL；Forulic acid 1.5-1500ng/mL；Tanshin polyphenolic acid B 10-3000ng/mL；The Radix Astragali
First glycosides 10-3000ng/mL；Formononetin 0.075-75ng/mL；Cryptotanshinone 0.075-75ng/mL；Tanshinone IIA 0.3-
300ng/mL；Serial standards mixed solution；
1.2.3 the preparation of inner mark solution: precision weighs Gardenoside 1.00mg, Sennoside B 1.00mg, Puerarin
1.00mg, chlorogenic acid 1.00mg, in 10mL volumetric flask, add proper amount of methanol and dissolve, and be settled to graduation mark, in being made into respectively
Mark Gardenoside, Sennoside B, Puerarin, the storing solution of chlorogenic acid.It is appropriate that precision pipettes each internal standard storing solution, with methanol dilution, joins
Make containing Gardenoside 1.00 μ g/mL-1, Sennoside B 1.00 μ g/mL, Puerarin 1.00 μ g/mL, chlorogenic acid 1.00 μ g/mL mixed
Close inner mark solution.
1.3 plasma sample pretreatment
Take plasma sample 100 μ L, the described mixing inner mark solution 10 μ L of accurate addition, formic acid 10 μ L, vortex 1min, add
Acetonitrile 400 μ L, vortex 1min, stand 2min, 14000rpm and be centrifuged 10min, take supernatant, and nitrogen is blown to do, and takes 100 μ L methyl alcohol multiple
Molten, vortex 60s, ultrasonic 60s, 14000rpm are centrifuged 10min, take supernatant 5 μ L sample introduction analysis.
1.4 confirmations analyzing method
Methodology in described rat plasma according to U.S. FDA biological sample analysis method guideline to analyze method
Methodology investigate, the degree of accuracy in method, precision, stability etc. determine high, medium and low three variable concentrations respectively
Three batch sample of rank.The investigation of the rate of recovery in the method degree of accuracy, is complete in 3 parallel sample of high, medium and low three concentration
Become；In withinday precision, day to day precision and study on the stability, 6 parts of test samples of the most parallel preparation are analyzed, real
The preci-sion and accuracy testing result represents with relative standard deviation RSD (%) and the degree of accuracy (%).It specifically includes following step
Rapid:
1.4.1 the specificity checking of method
Under the experiment testing conditions set up, prepare the blank blood sample of 6 different batches rats respectively (without to be analyzed
Thing and internal standard) it is analyzed, detection endogenous substance in plasma and other co-elute thing annoyance levels to target analytes, knot
The separating degree of fruit 11 target-finding compounds of display and 4 internal standard compositions is high, and peak shape is good, does without endogenous material in blood plasma
Disturb mensuration.Result is as shown in Figure 1.
Fig. 1 is the blood plasma chromatogram of the plasma sample measured, and wherein, (A) is blank plasma chromatogram；(B) it is blank blood
Slurry adds each compound and interior target chromatogram；(C) it is that rat is to the blood plasma chromatogram of 30min after 1.5g/kg cerebral ischemic capsule
Figure.
1.4.2 the calibration curve of plasma sample
Take 100 μ L blank plasmas, add mixing inner mark solution and each 10 μ L of series concentration hybrid standard product solution, formic acid 10
μ L, vortex 1min, add acetonitrile 400 μ L, vortex 1min, stands 2min, 14000rpm and is centrifuged 10min, takes supernatant, and nitrogen blows
To dry, taking 100 μ L methyl alcohol and redissolve, vortex 60s, ultrasonic 60s, 18000 × g are centrifuged 10min, take supernatant 10 μ L sample introduction and measure.
With determinand with internal standard peak area ratio (Y) as ordinate, with testing concentration (X) as abscissa, make equation of linear regression, meter
Calculation weight is 1/X2.Result is as shown in table 3.
Table 3 range of linearity and minimum detectable level (n=3)
As shown in Table 3, r is all higher than 0.9910, and the minimum quantitative limit of 11 kinds of compounds is all from 0.075-10ng/mL, table
Bright set up analysis method has higher sensitivity, can be used for the detection of low concentration medicine in blood plasma.
1.4.3 the precision of method and the degree of accuracy
Take 100 μ L blank plasmas, the operation pre-processed by above-mentioned plasma sample, be configured to containing standard concentration be low, in,
Each 6 parts of high plasma sample, measures, record testing compound peak area and corresponding internal standard peak area, calculates its ratio, substitute into
Column criterion curve calculates concentration, obtains recording concentration value.Precision is the variation value recording concentration, represents with RSD, the degree of accuracy
Represent with the percentage of the concentration recorded with actual concentration ratio, record mean ± SD that concentration is 6 results.In for three days on end
Parallel processing, measure 3 batches of plasma samples, respectively calculate batch in, batch between preci-sion and accuracy, result is as shown in table 4.
Table in 4 days with day to day precision (Intra-precision and Inter-precision, n=6)
As shown in Table 4, Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, Ah are measured while being set up
Acid Wei, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, the LC-MS/MS of tanshinone IIA analyze method and have preferably
The degree of accuracy and precision, meet Determination of Biological Samples precision requirement.
1.4.4 extraction recovery and matrix effect
Take blank plasma, add hybrid standard product solution 10 μ L, prepare high, normal, basic three concentration level simulating blood plasma samples
Each 6 parts of solution, the operation pre-processed by described plasma sample, sample introduction 10 μ L measures；Separately take blank plasma 100 μ L, accurate addition
Described mixing inner mark solution 10 μ L, formic acid 10 μ L, vortex 1min, add acetonitrile 400 μ L, vortex 1min, stand 2min,
18000g is centrifuged 10min, takes supernatant, adds high, normal, basic three horizontal hybrid standard product solution 10 μ L, and nitrogen is blown to do, and uses 100 μ
L methanol solution redissolves, the preparation compound standard solution containing bare substrate, and sample introduction 10 μ L measures, and records peak area；By front and back
Both peak area percent calculate the rate of recovery, the results are shown in Table 5, and three concentration extraction recovery mean values meet biological sample pair
Requirement in extraction recovery.
Take 100 μ L blank plasmas, the described mixing inner mark solution 10 μ L of accurate addition, formic acid 10 μ L, vortex 1min, add
Acetonitrile 400 μ L, vortex 1min, stand 2min, 18000g and be centrifuged 10min, take supernatant, adds high, normal, basic three levels mixing mark
Quasi-product solution 10 μ L, nitrogen is blown to do, and redissolves with 100 μ L methyl alcohol, and sample introduction 10 μ L measures, and records peak area；Prepare with methyl alcohol simultaneously
High, normal, basic three horizontal hybrid standard product solution, sample introduction 10 μ L measures, and records peak area；Various chemical combination are obtained by two kinds of methods
The percentage calculation matrix effect of thing peak area.Result is as shown in table 5.
Table 5 extraction recovery and matrix effect (n=6)
As shown in Table 5: all rate of recovery are all higher than 60%, show that the method for optimized acetonitrile precipitation albumen is suitable for greatly
The pre-treatment of mouse plasma sample；Though Paeoniflorin matrix effect between 127%-154% the most stable, other compound matrix
Effect, all between 75.3%-115%, shows that surveyed compound matrix effect is less, and sample substrate and altogether effluent are to institute
Survey compound without impact.
The determination of minimum quantitative limit (LLOQ)
Take blank plasma 100 μ L, the described mixing inner mark solution 10 μ L of accurate addition, formic acid 10 μ L, vortex 1min, add
Acetonitrile 400 μ L, vortex 1min, stand 2min, 14000rpm and be centrifuged 10min, take supernatant, and nitrogen is blown to do, and takes 100 μ L methyl alcohol multiple
Molten, vortex 60s, ultrasonic 60s, 18000g are centrifuged 10min, take supernatant 10 μ L sample introduction analysis.When S/N >=5, concentration is defined as
Minimum quantitative limit (LLOQ), then prepares 6 parts of samples, substitutes in retinue calibration curve and carries out calculating its veracity and precision,
Measurement result is shown in Table 3.
1.4.6 plasma sample study on the stability
Take blank plasma 100 μ L, be configured to the standard items plasma sample of basic, normal, high three concentration, investigate it respectively in room
Temperature 24h, multigelation 3 times and the stability of-20 DEG C of freeze thawing fortnights, the results are shown in Table 6.
Table 6 Stability Determination
As known from Table 6, each compound results RSD value all meets the requirements, and points out this laboratory sample should be complete within 2 week
Become sample determination, the storage of sample in experimentation, process the most stable, on analyze without conspicuousness impact.
1.5 discuss
The optimization of the phase that 1.5.1 flows
For reaching optimal separation state, the composition of flowing phase is optimized by the present inventor, and employing acetonitrile-
0.1% formic acid water carries out gradient elution, and testing compound abundance is higher, and sensitivity strengthens, the advantages such as analysis time is short.
1.5.2 the optimization of Mass Spectrometry Conditions
In order to improve response intensity and the sensitivity of each compound, this experiment source dates to each compound, mass spectrum
Parameter and MRM are optimized respectively, use the positive and negative mode simultaneously scanned, and the corresponding of each compound reaches more satisfactory shape
State.
1.5.3 interior target selects
Use interior target purpose partly in order to reduce not parallel, the Drug absorbability etc. of sample treatment to analysis result
On the other hand impact, is the error produced for difference because of Ionization Efficiency when reducing Mass Spectrometer Method.Reason should be selected as far as possible
Changing character, the chromatographic behavior material similar to determinand as internal standard, homologues or the structure of general many selection analytes are similar
Thing.According to above principle, we have selected Gardenoside, sennoside, Puerarin, chlorogenic acid respectively as internal standard.Through test, at this
Under conditions of experiment determines, all internal standards are the most relative the most stable, without endogenous with instrument with testing compound all retention times
Material disturbs.Therefore, we select above internal standard as the internal standard of all kinds of determinands.
1.5.4 the investigation of plasma sample processing method
In order to farthest remove removing protein and extract determinand, we remove method, sample and solvent ratio to albumen
Example, the addition volume of formic acid are investigated.
It was found that, use the acetonitrile precipitation of protein method than liquid-liquid extraction to the rate of recovery of analysis ingredient and matrix
The effect of effect is more preferable, on conventional experiment basis, finds that sample and acetonitrile sedimentation effect under the ratio of 1:4 is preferable, sample
The rate of recovery is preferable, adds formic acid and is added without formic acid and has a certain impact sample recovery rate.First has been investigated in this experiment simultaneously
The volume (0,5,10 and 20) that acid the adds impact on experiment, result shows, when adding 10 μ L formic acid, experimental result is preferable.Warp
Consult pertinent literature, it is contemplated that the advantages such as high treating effect, low cost, processing method are simple, therefore use acetonitrile precipitation albumen side
Plasma sample is processed by method.
Embodiment 2, the pharmacokinetics of gastric infusion rat hindbrain Xintong capsule main component
The collection of 2.1 plasma samples
Subject male SD rat pre-adaptation one week, can't help water 12h in test fasting the night before last, and second day by animal used as test
Be randomly divided into 2 groups, often group 8, press 0.5g/kg and 1.5g/kg dosage gastric infusion respectively, in administration after 0.083,0.167,
0.25,0.5,0.75,1,1.5,2,4,8,12 and 24h eyeground vein clump takes blood, is placed in heparin centrifuge tube, and 8000g is centrifuged
10min, takes out upper plasma, in-20 DEG C of Refrigerator stores, to be measured.
2.2 plasma sample pretreatment
Take plasma sample 100 μ L, accurate addition internal standard mixed solution 10 μ L, formic acid 10 μ L, vortex 1min, add acetonitrile
400 μ L, vortex 1min, stand 2min, 18000g and be centrifuged 10min, take supernatant, and nitrogen dries up, and redissolves with 100 μ L methyl alcohol, vortex
60s, ultrasonic 60s, 18000g are centrifuged 10min, take supernatant 10 μ L sample introduction.
2.3 rat plasma sample analyses
The plasma sample obtained in the acquisition step of described plasma sample is pre-processed, uses in embodiment 1 and built
Vertical LC-MS/MS condition is measured.The final caffeic acid, forulic acid, formononetin, hidden of only detecting in plasma sample
Tanshinone, five compositions of tanshinone IIA.When measuring plasma sample, analysis method is carried out quality control, has set up with rower
Directrix curve, and QC (Quality Control) sample of high, medium and low three concentration of accompanying.
Plasma sample carries out LC-MS analysis, with accompanying calibration curve calculate content, with time m-blood concentration mapping, paint
Caffeic acid processed, forulic acid, formononetin, Cryptotanshinone, the blood concentration-time curve map of tanshinone IIA.
Fig. 2-Fig. 6 is the blood concentration-time curve of each composition in rat plasma, caffeic acid during wherein a is rat plasma
Blood concentration-time curve；B is the blood concentration-time curve of forulic acid in rat plasma；C is awns handle in rat plasma
The blood concentration-time curve of xanthin；D is the blood concentration-time curve of Cryptotanshinone in rat plasma；E is rat serum
The blood concentration-time curve of tanshinone IIA in slurry.
Knowable to Fig. 2-Fig. 6, after Oral Administration in Rats cerebral ischemic capsule, caffeic acid, forulic acid, formononetin, Cryptotanshinone,
Tanshinone IIA all can absorbed into serum, caffeic acid, forulic acid, formononetin have absorption fast and eliminate fast, Cryptotanshinone, pellet
Ginseng ketone II A has absorption fast and eliminates the features such as slower；Simultaneously along with the increase of dosage, five kinds of compound AUC also become big, table
Bright its absorbs and increases.
2.4 rats are administered the pharmacokinetic parameter after cerebral ischemic capsule
The blood concentration-time data of rat after being administered are processed, according to minimum Akaike ' s with DAS 1.0 software
Information criterion (AIC) information judges, caffeic acid, forulic acid, formononetin, Cryptotanshinone, Tanshinone II
A pharmacokinetic model meets one compartment open model.Use statistics moments method simultaneously, calculate its main pharmacokinetic parameter, institute's medication
Dynamic software of learning is DAS Statistics Version 1.0 (Mathematical Pharmacology Professional
Committee of China, Shanghai, China), result is as shown in table 7.
Table 7 rat is administered the pharmacokinetic parameter of each main component after cerebral ischemic capsule
The above results shows, the liquid chromatography tandem mass spectrometry detection plasma sample midbrain Xintong capsule that the present invention is set up
Main component Paeoniflorin, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, awns handle
Xanthin, Cryptotanshinone, the method for tanshinone IIA, meet vivo biodistribution sample determination requirement, and method sensitivity is good, specificity
By force, stable, reliable, and can apply to pharmacokinetics after cerebral ischemic capsule oral is administered.
Claims (4)
1. the method simultaneously detecting blood plasma midbrain Xintong capsule main component, described cerebral ischemic capsule main component includes Chinese herbaceous peony
Medicine glycosides, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, hidden
Tanshinone, tanshinone IIA, described method includes: the preparation of step (1) sample and step (2) use Liquid Chromatography-Tandem Mass Spectrometry
Method detects；
The method that the preparation employing of described step (1) sample comprises the following steps:
A) in test plasma sample, add mixing inner mark solution, formic acid, and mix；It is designated as Cape jasmine in described mixing inner mark solution
Sub-glycosides, Sennoside B, Puerarin and chlorogenic acid；
B) add acetonitrile, stand after mixing；
C) centrifugal, collect supernatant, nitrogen dries up, and collects dried object；
D) the dried object methyl alcohol obtained is redissolved, mix, be centrifuged, take supernatant；
Described step (2) detection liquid phase chromatogram condition used is as follows:
Chromatographic column is C18Post；
Flowing is that 0.1% aqueous formic acid forms by acetonitrile and volume fraction；
Type of elution is gradient elution；
The program of described gradient elution is as follows:
It is constant that the volume fraction of 0-10min: acetonitrile maintains 30%；
The volume fraction of 10-15min: acetonitrile is increased to 80% by 30%；
The volume fraction of 15-20min: acetonitrile is increased to 95% by 80%；
It is constant that the volume fraction of 20-30min: acetonitrile maintains 95%；
In described liquid phase chromatogram condition, chromatographic column is Agilent Eclipse Plus C18Post, specification is: 4.6mm × 100mm,
A diameter of 1.8 μm of filler；Flow rate of mobile phase is 300 μ L/min；Column temperature is 30 DEG C；Sample size is 10 μ L；
Described step (2) detection Mass Spectrometry Conditions used is as follows: use API 3200TMLC/MS/MS liquid matter analyzes system；Ion gun, negative ions is switched fast analytical model, multiple-reaction monitoring scan mode；
Described negative ions is switched fast in analytical model, and positive ion mode parameter is set to: gas curtain gas (CUR): 10psi；Touch
Hit gas (CAD): 8psi；Ion spray voltage (IS): 5000V；Temperature (TEM): 500 DEG C；Air-flow 1 (GS1): 40psi；Air-flow 2
(GS2): 30psi；Heating (Ihe): On；
Negative ion mode parameter is set to: gas curtain gas (CUR): 20psi；Collision gas (CAD): 8psi；Ion spray voltage
(IS) :-4500V；Temperature (TEM): 500 DEG C；Air-flow 1 (GS1): 30psi；Air-flow 2 (GS2): 30psi；Heating (Ihe): On；
In described Mass Spectrometry Conditions, described multiple-reaction monitoring method parameter is as follows:
Paeoniflorin: parent ion mass number (Q1): 478.6；Fragment ion masses number (Q3): 121.1；Solve bunch voltage (DP) :-45V；
Entrance potential (EP) :-4V；Sampling time (Dwell Time): 100；Impact energy :-30V；Collision cell exit potential (CXP) :-
1V；Retention time (Rt): 4.47min；
β ecdysterone: parent ion mass number (Q1): 478.9；Fragment ion masses number (Q3): 159.0；Solve bunch voltage (DP) :-
60V；Entrance potential (EP) :-10V；Sampling time (Dwell Time): 100；Impact energy :-37V；Collision cell exit potential
(CXP) :-1V；Retention time (Rt): 4.48min；
Amarogentin: parent ion mass number (Q1): 455.8；Fragment ion masses number (Q3): 322.8；Solve bunch voltage :-50V；Enter
Mouth voltage (EP) :-4.5V；Sampling time (Dwell Time): 100；Impact energy :-20V；Collision cell exit potential (CXP) :-
22V；Retention time (Rt): 4.49min；
Mulberroside A: parent ion mass number (Q1): 567.0；Fragment ion masses number (Q3): 242.6；Solve bunch voltage: 60V；Entrance
Voltage (EP): 10V；Sampling time (Dwell Time): 100；Impact energy: 38V；Collision cell exit potential (CXP): 16V；Retain
Time (Rt): 4.53min；
Caffeic acid: parent ion mass number (Q1): 178.9；Fragment ion masses number (Q3): 134.9；Solve bunch voltage: 35V；Entrance
Voltage (EP): 3.5V；Sampling time (Dwell Time): 100；Impact energy: 24V；Collision cell exit potential (CXP): 0.5V；Protect
Stay time (Rt): 5.28min；
Forulic acid: parent ion mass number (Q1): 192.8；Fragment ion masses number (Q3): 133.9；Solve bunch voltage :-31V；Entrance
Voltage (EP) :-4V；Sampling time (Dwell Time): 100；Impact energy :-23V；Collision cell exit potential (CXP) :-1V；Protect
Stay time (Rt): 7.32min；
Tanshin polyphenolic acid B: parent ion mass number (Q1): 716.5；Fragment ion masses number (Q3): 518.5；Solve bunch voltage :-55V；Enter
Mouth voltage (EP) :-4.5V；Sampling time (Dwell Time): 100；Impact energy :-25V；Collision cell exit potential (CXP) :-
40V；Retention time (Rt): 8.92min；
Astragaloside IV: parent ion mass number (Q1): 807.0；Fragment ion masses number (Q3): 807.0；Solve bunch voltage: 140V；Enter
Mouth voltage (EP): 10V；Sampling time (Dwell Time): 100；Impact energy: 50V；Collision cell exit potential (CXP): 55V；Protect
Stay time (Rt): 18.79min；
Formononetin: parent ion mass number (Q1): 267.0；Fragment ion masses number (Q3): 251.8；Solve bunch voltage :-16V；
Entrance potential (EP) :-2V；Sampling time (Dwell Time): 100；Impact energy :-27V；Collision cell exit potential (CXP) :-
5V；Retention time (Rt): 20.43min；
Cryptotanshinone: parent ion mass number (Q1): 297.0；Fragment ion masses number (Q3): 254.2；Solve bunch voltage: 50V；Enter
Mouth voltage (EP): 9V；Sampling time (Dwell Time): 100；Impact energy: 35V；Collision cell exit potential (CXP): 15V；Protect
Stay time (Rt): 25.64min；
Tanshinone IIA: parent ion mass number (Q1): 295.2；Fragment ion masses number (Q3): 277.1；Solve bunch voltage: 58V；Enter
Mouth voltage (EP): 10V；Sampling time (Dwell Time): 100；Impact energy: 27V；Collision cell exit potential (CXP): 21V；Protect
Stay time (Rt): 27.53min；
Gardenoside (IS): parent ion mass number (Q1): 387.0；Fragment ion masses number (Q3): 224.7；Solve bunch voltage :-40V；
Entrance potential (EP) :-9V；Sampling time (Dwell Time): 100；Impact energy :-15V；Collision cell exit potential (CXP) :-
15V；Retention time (Rt): 4.47min；
Sennoside B (IS): parent ion mass number (Q1): 860.6；Fragment ion masses number (Q3): 385.9；Solve bunch voltage :-
87V；Entrance potential (EP) :-10V；Sampling time (Dwell Time): 100；Impact energy :-60V；Collision cell exit potential
(CXP) :-2V；Retention time (Rt): 4.43min；
Puerarin (IS): parent ion mass number (Q1): 414.8；Fragment ion masses number (Q3): 266.9；Solve bunch voltage :-50V；
Entrance potential (EP) :-9V；Sampling time (Dwell Time): 100；Impact energy :-46V；Collision cell exit potential (CXP) :-
2V；Retention time (Rt): 4.44min；
Chlorogenic acid (IS): parent ion mass number (Q1): 352.8；Fragment ion masses number (Q3): 190.9；Solve bunch voltage :-33V；
Entrance potential (EP) :-4V；Sampling time (Dwell Time): 100；Impact energy :-32V；Collision cell exit potential (CXP) :-
2V；Retention time (Rt): 4.45min.
2. a liquid chromatography tandem mass spectrometry detects Paeoniflorin in plasma sample, β ecdysterone, amarogentin, mulberry skin simultaneously
Glycosides A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, the method for content of tanshinone IIA,
Comprise the steps:
(1), the preparation of calibration curve:
Take the Paeoniflorin of a series of concentration, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Huang
Stilbene first glycosides, formononetin, Cryptotanshinone, the hybrid standard product solution of tanshinone IIA add in blank plasma samples, according to power
Profit requires that the sample preparation methods described in 1 is prepared, then to the supernatant obtained according to the liquid phase look described in claim 1
Spectrum tandem mass spectrometry detects, and records the Paeoniflorin of each concentration, β ecdysterone, amarogentin, Mulberroside A, coffee respectively
Coffee acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, peak area that tanshinone IIA is corresponding；
With Paeoniflorin and internal standard peak area ratio Y as ordinate, with Paeoniflorin concentration X as abscissa, prepare the linear of Paeoniflorin
Regression equation；
With β ecdysterone and internal standard peak area ratio Y as ordinate, with β ecdysterone concentration X as abscissa, preparation β is casted off a skin steroid
The equation of linear regression of ketone；
With amarogentin and internal standard peak area ratio Y as ordinate, with amarogentin concentration X as abscissa, prepare amarogentin
Equation of linear regression；
With Mulberroside A and internal standard peak area ratio Y as ordinate, with Mulberroside A concentration X as abscissa, prepare the line of Mulberroside A
Property regression equation；
With caffeic acid and internal standard peak area ratio Y as ordinate, with caffeic acid concentration X as abscissa, prepare caffeinic linearly
Regression equation；
With forulic acid and internal standard peak area ratio Y as ordinate, with forulic acid concentration X as abscissa, prepare the linear of forulic acid
Regression equation；
With tanshin polyphenolic acid B and internal standard peak area ratio Y as ordinate, with tanshin polyphenolic acid B concentration X as abscissa, prepare the line of tanshin polyphenolic acid B
Property regression equation；
With Astragaloside IV and internal standard peak area ratio Y as ordinate, with Astragaloside IV concentration X as abscissa, prepare Astragaloside IV
Equation of linear regression；
With formononetin and internal standard peak area ratio Y as ordinate, with formononetin concentration X as abscissa, prepare awns handle
The equation of linear regression of xanthin；
With tanshinone IIA and internal standard peak area ratio Y as ordinate, with tanshinone IIA concentration X as abscissa, prepare tanshinone
The equation of linear regression of II A；
With Cryptotanshinone and internal standard peak area ratio Y as ordinate, with Cryptotanshinone concentration X as abscissa, prepare Cryptotanshinone
Equation of linear regression；
(2), Paeoniflorin in test plasma sample, β ecdysterone, amarogentin, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B,
Astragaloside IV, formononetin, Cryptotanshinone, the assay of tanshinone IIA:
Test plasma sample is prepared according to the sample preparation methods described in claim 1, then to the supernatant obtained
Detect according to the liquid chromatography tandem mass spectrometry described in claim 1, and record Paeoniflorin, β ecdysterone, bitter apricot respectively
Benevolence glycosides, Mulberroside A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, tanshinone IIA are corresponding
Peak area；
With Paeoniflorin and internal standard peak area ratio Y as ordinate, with Paeoniflorin concentration X as abscissa, prepare the linear of Paeoniflorin
Regression equation；
With β ecdysterone and internal standard peak area ratio Y as ordinate, with β ecdysterone concentration X as abscissa, preparation β is casted off a skin steroid
The equation of linear regression of ketone；
With amarogentin and internal standard peak area ratio Y as ordinate, with amarogentin concentration X as abscissa, prepare amarogentin
Equation of linear regression；
With Mulberroside A and internal standard peak area ratio Y as ordinate, with Mulberroside A concentration X as abscissa, prepare the line of Mulberroside A
Property regression equation；
With caffeic acid and internal standard peak area ratio Y as ordinate, with caffeic acid concentration X as abscissa, prepare caffeinic linearly
Regression equation；
With forulic acid and internal standard peak area ratio Y as ordinate, with forulic acid concentration X as abscissa, prepare the linear of forulic acid
Regression equation；
With tanshin polyphenolic acid B and internal standard peak area ratio Y as ordinate, with tanshin polyphenolic acid B concentration X as abscissa, prepare the line of tanshin polyphenolic acid B
Property regression equation；
With Astragaloside IV and internal standard peak area ratio Y as ordinate, with Astragaloside IV concentration X as abscissa, prepare Astragaloside IV
Equation of linear regression；
With formononetin and internal standard peak area ratio Y as ordinate, with formononetin concentration X as abscissa, prepare awns handle
The equation of linear regression of xanthin；
With tanshinone IIA and internal standard peak area ratio Y as ordinate, with tanshinone IIA concentration X as abscissa, prepare tanshinone
The equation of linear regression of II A；
With Cryptotanshinone and internal standard peak area ratio Y as ordinate, with Cryptotanshinone concentration X as abscissa, prepare Cryptotanshinone
Equation of linear regression.
Method the most according to claim 1 and 2, it is characterised in that: in step a), described test plasma sample and formic acid
Volume ratio is 100:10；
In step b), the time of described standing is 2-5min；Described test plasma sample is 1:4 with the volume ratio of acetonitrile；
Step a), b), c) and d) in, described mixing all uses the mode of vortex, and the time of vortex is 1-2min；
Step c) and d) in, described centrifugal condition is 18000 × g-19000 × g and is centrifuged 10-15min.
4. Paeoniflorin, β ecdysterone, amarogentin, the mulberry skin in measuring cerebral ischemic capsule of the method described in claim 1 or 2
Glycosides A, caffeic acid, forulic acid, tanshin polyphenolic acid B, Astragaloside IV, formononetin, Cryptotanshinone, the pharmacokinetics of tanshinone IIA
In application.
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| CN105241980B (en) * | 2015-11-12 | 2017-05-24 | 陕西步长制药有限公司 | Rapid separation liquid chromatography detection method for naoxintong capsules |
| CN107957452A (en) * | 2016-10-17 | 2018-04-24 | 天士力医药集团股份有限公司 | A kind of method of phenolic acid in detection blood plasma |
| CN107422052B (en) * | 2017-06-29 | 2020-06-05 | 天津中医药大学 | Green extraction and determination method for various compounds in Chinese herbal compound Naoxintong |
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| CN108982703B (en) * | 2018-08-23 | 2021-05-14 | 中国检验检疫科学研究院 | Liquid chromatography-mass spectrometry detection method for polyphenol substances |
| CN109030664A (en) * | 2018-09-27 | 2018-12-18 | 湖北科技学院 | The method for measuring the content of 7 kinds of polyphenol active ingredients in different cultivars sweet osmanthus simultaneously by HPLC-MS-MS method |
| CN109856267B (en) * | 2019-01-23 | 2022-02-18 | 牡丹江友搏药业有限责任公司 | Method for simultaneously determining contents of multiple components in Chinese medicinal preparation based on LC-MS technology |
| CN110231424B (en) * | 2019-07-23 | 2021-12-14 | 南京中医药大学 | Method for simultaneously and quantitatively detecting main components of Shuangshen Ping lung granules in blood plasma |
| CN110554131A (en) * | 2019-09-29 | 2019-12-10 | 湖北神农汉方生物科技有限公司 | Quality detection method for effective components of novel traditional Chinese medicine decoction |
| CN112595787A (en) * | 2020-11-27 | 2021-04-02 | 山东大学 | Detection method and application of paeoniflorin, calycosin glycoside and amygdalin in plasma |
| CN113192649B (en) * | 2021-06-01 | 2023-09-22 | 山东英盛生物技术有限公司 | System for guiding individual accurate medication of epileptic diseases |
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