CN109239224A - 9 kinds of method for quantitatively determining while enter blood component in semen ziziphi spinosae water extract - Google Patents
9 kinds of method for quantitatively determining while enter blood component in semen ziziphi spinosae water extract Download PDFInfo
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Abstract
The invention belongs to be metabolized studying technological domain in Chinese patent drug body, enters the low overlong time of HPLC-UV sensitivity in blood component quantitative analysis to solve current semen ziziphi spinosae, be not suitable for micro constitutent in analysis blood plasma；LC-MS/MS is only used for the pharmacokinetics of measurement single component, the problem of being unfavorable for the research of Pharmacokinetics, provides in a kind of semen ziziphi spinosae water extract 9 kinds of method for quantitatively determining while enter blood component.Enter blood component with 9 kinds in UHPLC-Q Exactive Orbitrap HR/MS Simultaneous Determination semen ziziphi spinosae water extract；Detection sensitivity is improved, analysis time is shortened；Realize quantitative analysis while to three kinds of flavones, saponin(e and alkaloids polar compounds；Analytical technology guarantee is provided in the intracorporal pharmacokinetic of rat model of normally having a sleepless night with PCPA for semen ziziphi spinosae, provides reference for other oral traditional Chinese medicine multicomponent Simultaneous Quantitative Analysis.
Description
Technical field
The invention belongs to be metabolized studying technological domain in Chinese patent drug body, and in particular to enter for 9 kinds in a kind of semen ziziphi spinosae water extract
Method for quantitatively determining while blood component.
Background technique
Semen ziziphi spinosae is rhamnaceae plant wild jujubeZiziphus jujuba Mill. var. spinosa (Bunge) Hu
The dry mature seed of ex H. F. Chou has nourishing heart tonifying liver, and antitoxic heart-soothing and sedative, arrest sweating is clinically to control the effect of promoting the production of body fluid
Treat the choice drug of insomnia.Modern pharmacological studies have shown that total saposins, general flavone, total alkaloid are that it plays calmness in semen ziziphi spinosae
The main active of hypnosis.
It is well known that effective component of chinese medicine absorbed into serum is the premise that its curative effect plays, and multicomponent, multiple target point, make more
It is its clinical effective feature with approach.Existing fitochemical studies show chemical component structure-rich multiplicity in semen ziziphi spinosae,
With saponins, flavonoids and alkaloids for its main characteristic component.The current pharmacokinetic studies in relation to semen ziziphi spinosae mainly collect
In in the research for giving a certain monomer component in rat semen ziziphi spinosae or semen ziziphi spinosae chromocor extract etc. by oral or intravenous.
However, administration form of the semen ziziphi spinosae water extract Oral administration closer to clinic.And semen ziziphi spinosae rich in flavonoids, saponins,
The chemical component of the various structures type such as alkaloids and triterpene acids, and the big phase of Pharmacokinetic Characteristics of different type ingredient
It is very unlike.It is infeasible in traditional Chinese medicine research with the pharmacokinetics behavior that a kind of ingredient represents a herb.In addition, at this stage in relation to wild jujube
The quantitative analysis that benevolence enters blood component mainly passes through HPLC-UV and LC-MS/MS method measurement spinosin and its derivative
6'''The pharmacokinetics of several compounds such as asafoetide acyl spinosin and Saponin A, Spine Date Seed jujubosideB.Point of HPLC-UV
The micro constitutent that sensitivity is low, in unsuitable analysis blood plasma is analysed, and analysis time is too long；Although LC-MS/MS measurement sensitivity
Height, but be only applied to determine the pharmacokinetics of single component.
Summary of the invention
The present invention is in order to solve the sensitivity for analysis that related semen ziziphi spinosae at this stage enters HPLC-UV in blood component quantitative analysis
It is low, be not suitable for the micro constitutent in analysis blood plasma, and analysis time is too long；Although LC-MS/MS measurement sensitivity is high, only answer
The problem of for determining the pharmacokinetics of single component, being unfavorable for the research of Pharmacokinetics, provides a kind of semen ziziphi spinosae water and mentions
9 kinds of method for quantitatively determining while enter blood component in object, and give normal and PCPA and have a sleepless night mould semen ziziphi spinosae water extract is oral
Application in the pharmacokinetics of type rat.
The present invention is realized by following technical solution: 9 kinds of quantitative determinations while enter blood component in a kind of semen ziziphi spinosae water extract
Method is both 9 kinds of work in quantitative determination semen ziziphi spinosae water extract using UHPLC-Q Exactive Orbitrap HR/MS method
Property ingredient: coclaurine, magnoflorine, Wei Caining, spinosin, swertisin, Kaempferol -3-ORutinoside, 6'''Asafoetide
Acyl spinosin, Saponin A and Spine Date Seed jujubosideB；Specific step is as follows:
(1) preparation of single reference substance stock solution: UHPLC-Q Exactive is added in accurately weighed reference substance respectively
The chromatography initial liquid phase of Orbitrap HR/MS analysis, is prepared into 2.032 mg/mL of coclaurine, 1.130 mg/ of magnoflorine
The single reference substance stock solution of mL, addition methanol are prepared into dimension and adopt peaceful 2.184 mg/mL, 2.016 mg/mL of spinosin, work as medicine
1.972 mg/mL of flavine, Kaempferol -3-O2.058 mg/mL of rutinoside, 6'''2.012 mg/ of asafoetide acyl spinosin
The single reference substance stock solution of mL, 2.040 mg/mL of Saponin A, 2.064 mg/mL of Spine Date Seed jujubosideB；
The preparation of working solution: it is molten that mixing reference substance is made in the above-mentioned single reference substance stock solution addition methanol of accurate measurement respectively
Liquid, in which: 325.12 ng/mL of coclaurine, 18080 ng/mL of magnoflorine, Wei Caining, 2419.2 ng/mL of spinosin, when
94.66 ng/mL of medicine flavine, Kaempferol -3-O246.96 ng/mL of rutinoside, 6'''Asafoetide acyl spinosin 128.77
Ng/mL, 652.8 ng/mL of Saponin A, 412.8 ng/mL of Spine Date Seed jujubosideB；Then accurate to measure above-mentioned mixing pair
According to product solution, respectively plus methanol is diluted to the 4/5 of mixed reference substance solution concentration, 1/2,1/4,1/8,1/40,1/80 times step by step,
It shakes up to get the working solution of mass concentration gradient；
(2) preparation of inner mark solution: select palmatin hydrochloride, daidzin, Astragaloside IV respectively as alkaloids, flavonoids,
The internal standard compound of saponins compound assay, accurately weighed internal standard compound, adds methanol to be prepared into palmatin hydrochloride 0.984 respectively
The single inner mark solution of mg/mL, 1.080 mg/mL of daidzin, 1.392 mg/mL of Astragaloside IV；It is molten that precision draws single internal standard
Hydrochloric 78.72 ng/mL of palmatine, 216.00 ng/mL of daidzin, Astragaloside IV 556.80 is made with methanol dilution in liquid
The mixing inner mark solution of ng/mL；
(3) foundation of standard curve: precision draws 100 μ L of blank plasma, and 10 μ L internal standards are added, and be vortexed 30 s, then adds respectively
Enter 10 μ L hybrid working solution, 30 s of vortex, solvent 5 min of vortex is then added, 13000 rpm, 4 DEG C of 10 min of centrifugation take
350 μ L of supernatant is added in clean EP pipe, and 1400 rpm traditional vacuums, 40 DEG C of air blow dryings, residue add 100 μ L initial
Mobile phase vortex 2 min, 13000 rpm, 4 DEG C of 5 min of centrifugation, take supernatant to carry out UHPLC-Q Exactive Orbitrap
HR/MS analysis；Using the ratio of Component peak area to be measured and internal standard peak area as ordinate (y), be added in blood plasma it is to be measured at
Divide the concentration of reference substance as abscissa (x), using 1/c2Weighted least-squares method carries out linear regression, establishes curve；
(4) detection of sample:
(a) preparation of semen ziziphi spinosae water extract: 500 g of semen ziziphi spinosae medicine materical crude slice is crushed to 60% and crosses No. 1 pharmacopeia sieve, adds 10 times of amount water, soak
30 min are steeped, are heated to boiling, 2 h of refluxing extraction is filtered, and filter residue adds 8 times of amount water again, and 2 h of heating and refluxing extraction is filtered, and is merged
Filtrate；It is concentrated under reduced pressure into crude drug meter concentration as 0.5 g/mL, freeze-drying, the used time is dissolved with water；
(b) acquisition of plasma sample: Oral Administration in Rats semen ziziphi spinosae water extract with crude drug meter concentration be 40 g/Kg, after administration in
To rat 300 μ L of blood sampling during 0.083-24 h, it is placed in the centrifuge tube containing 0.1% sodium heparin anticoagulant, stands 30 min,
4 DEG C, 3500 rpm, 10 min of centrifugation, separated plasma set -20 DEG C of preservations, for use；
(c) detection of sample: precision draws 100 μ L of rat plasma, is handled using the processing method of blank plasma samples,
For the solvent used in plasma sample processing for acetonitrile, dosage is 3 times of plasma sample；The plasma containing drug sample handled well is carried out
UHPLC-Q Exactive Orbitrap HR/MS analysis, the concentration of 9 kinds of ingredients in plasma sample is calculated according to standard curve.
The chromatographic condition of the UHPLC-Q Exactive Orbitrap HR/MS analysis: chromatographic column ACQUITY
UPLC HSS T3 column, the mm of 150 mm × 2.1,1.8 μm, mobile phase A: B is acetonitrile: 0.1% formic acid water or A:B are 0.1% first
Sour acetonitrile: 0.1% formic acid water, gradient elution program are 0~1.5 min, 17% A；1.5~3 min, 17 → 19% A；3~7
Min, 19 → 33% A；7~12 min, 33 → 98% A；12~14 min, 98 → 17% A；14~19 min, 17% A；Flow velocity
For 0.3 mL/min；Column temperature is 40 DEG C；Sample volume is 3 μ L；
Mass Spectrometry Conditions: ion source: the source HESI；Positive and negative ion switches while scanning；Operating mode: Full-MS；Mass spectrometry parameters: sheath
Gas velocity: 35 arb；Secondary air speed: 10 arb；Spray voltage: 3.2 KV(+), 2.5 KV(-)；Capillary temperature: 320
℃；Ion source temperature: 300 DEG C；Resolution ratio 70 000；Scanning range m/z 150-1500 Da.
The chromatography initial liquid phase of the analysis of UHPLC-Q Exactive Orbitrap HR/MS described in step (1) is 17%
0.1% formic acid acetonitrile -83% 0.1% formic acid water.
In the step (b) respectively rat administration after 0.083,0.167,0.333,0.5,0.75,1,2,4,6,10,
12, blood was collected to rat by 24 h.
The present invention selects internal standard method to carry out 9 kinds of ingredient simultaneous quantitatives, since ingredient to be measured adheres to 3 variety classes separately, therefore divides
Do not have chosen 3 kinds of different types of internal standards, palmatin hydrochloride, daidzin, Astragaloside IV respectively as alkaloids, flavonoids,
The internal standard of saponins compound assay.
The solvent used in the blank plasma samples processing is used for any one in methanol, acetonitrile or ethyl acetate
Amount is 3-5 times of blank plasma samples.Preferably, for acetonitrile, dosage is the solvent used in the blank plasma samples processing
3 times of blank plasma samples.
When mobile phase is investigated, mobile phase A in chromatographic condition: B is preferably 0.1% formic acid acetonitrile: 0.1% formic acid water.
The present invention has carried out reasonable setting to the selection of chromatographic condition, internal standard substance and sample pre-treatments condition, improves
Detection sensitivity, shortens analysis time；It realizes to three kinds of flavonoids, saponins and alkaloids opposed polarity compounds
While quantitative analysis；Can be used for semen ziziphi spinosae water extract oral administration after normally with PCPA insomnia rat model blood plasma in 9 kinds at
The pharmacokinetic studies divided；Technical Reference is provided for other oral traditional Chinese medicine multicomponent Simultaneous Quantitative Analysis.
To sum up, with 9 kinds of (coclaurine, magnoflorine, Wei Caining, spinosin, swertisin, mountains in semen ziziphi spinosae water extract
How phenol -3-ORutinoside, 6'''Asafoetide acyl spinosin, Saponin A and Spine Date Seed jujubosideB) active constituent be research
Object establishes in the completely new semen ziziphi spinosae water extract body of one kind 9 kinds using UHPLC-Q Exactive Orbitrap HR/MS technology
Quantitative analysis method while active constituent.9 kinds of ingredients selected by the present invention cover the activity that 3 class polarity are different in semen ziziphi spinosae
Ingredient, so that the pharmacokinetic of semen ziziphi spinosae is more fully.Full-MS scan pattern is smart by the level-one of high-performance parent ion
True mass number is used as quota ion, realizes after semen ziziphi spinosae water extract oral administration rat that 9 kinds enter blood component in single analysis together
Shi Jinhang accurate quantitative analysis.
Detailed description of the invention
Fig. 1 is the extraction ion flow graph of 9 kinds of ingredients, in figure, and: A is blank plasma, and B is blank plasma+reference substance, C be containing
Medicine blood plasma；Fig. 2 be normal group (C) and model group (M) Oral Administration in Rats semen ziziphi spinosae water extract after in 9 kinds of ingredients average drug it is dense
Degree-time graph.
Specific embodiment
The present invention is made further to illustrate in detail, completely below with reference to embodiment.The reagent used in each embodiment
It is as follows with equipment:
1. instrument: Thermo fisher U3000 Ultra Performance Liquid Chromatography instrument configures on-line degassing machine, quaternary gradient pump, column temperature
Case, UV detector, autosampler (Thermo Fisher Scientific company of the U.S.), Thermo ScientificTM
Q ExactiveTMOrbitrap mass spectrograph (Germany)；Ten a ten thousandth assay balance of CPA225D type, German Sartorius
Beijing instrument system Co., Ltd；Traditional vacuum concentrating instrument, German Eppendorf company.
2. drug and reagent: reference substance: coclaurine (lot number HC225036198), magnoflorine (lot number
HA061308198), Wei Caining (lot number HV187847198), spinosin (lot number 20160314), swertisin (lot number
HA062908198), Kaempferol -3-O-Rutinoside (lot number HK201623198), 6'''Asafoetide acyl spinosin (lot number
20160313), Saponin A (20160315 A of lot number), daidzin (lot number HD031189198), Astragaloside IV (lot number
HA012079198 it) is purchased from Baoji time Biotechnology Co., Ltd, Spine Date Seed jujubosideB (lot number 20170210) is purchased from south
Capital spring and autumn bioengineering Co., Ltd, palmatin hydrochloride (lot number 130427) are purchased from the limited public affairs of Sichuan Province's Wei Keqi biotechnology
Department, the mass fraction through HPLC normalization method measurement reference substance are all larger than 98%.Fenclonine (PCPA) (lot number GK01-
JBQH), purchased from uncommon love (chemical conversion) the industrial development Co., Ltd of ladder, purity is greater than 98%.Acetonitrile, formic acid are that mass spectrum is pure, are purchased from
Fisher company, methanol are that mass spectrum is pure, are purchased from Merck & Co., Inc..Other reagents are that analysis is pure.
Embodiment 1: 9 kinds of method for quantitatively determining while enter blood component in a kind of semen ziziphi spinosae water extract, using UHPLC-Q
Exactive Orbitrap HR/MS method is both 9 kinds of active constituents in quantitative determination semen ziziphi spinosae water extract: coclaurine, lily magnolia
Flower alkali, Wei Caining, spinosin, swertisin, Kaempferol -3-ORutinoside, 6'''Asafoetide acyl spinosin, semen ziziphi spinosae
Saponin A and Spine Date Seed jujubosideB；Specific step is as follows:
(1) preparation of single reference substance stock solution: UHPLC-Q Exactive is added in accurately weighed reference substance respectively
The chromatography initial liquid phase of Orbitrap HR/MS analysis, is prepared into 2.032 mg/mL of coclaurine, 1.130 mg/ of magnoflorine
The single reference substance stock solution of mL, addition methanol are prepared into dimension and adopt peaceful 2.184 mg/mL, 2.016 mg/mL of spinosin, work as medicine
1.972 mg/mL of flavine, Kaempferol -3-O2.058 mg/mL of rutinoside, 6'''2.012 mg/ of asafoetide acyl spinosin
The single reference substance stock solution of mL, 2.040 mg/mL of Saponin A, 2.064 mg/mL of Spine Date Seed jujubosideB；
The preparation of working solution: it is molten that mixing reference substance is made in the above-mentioned single reference substance stock solution addition methanol of accurate measurement respectively
Liquid, in which: 325.12 ng/mL of coclaurine, 18080 ng/mL of magnoflorine, Wei Caining, 2419.2 ng/mL of spinosin, when
94.66 ng/mL of medicine flavine, Kaempferol -3-O246.96 ng/mL of rutinoside, 6'''Asafoetide acyl spinosin 128.77
Ng/mL, 652.8 ng/mL of Saponin A, 412.8 ng/mL of Spine Date Seed jujubosideB；Then accurate to measure above-mentioned mixing pair
According to product solution, respectively plus methanol is diluted to the 4/5 of mixed reference substance solution concentration, 1/2,1/4,1/8,1/40,1/80 times step by step,
It shakes up to get the working solution of mass concentration gradient；
(2) preparation of inner mark solution: select palmatin hydrochloride, daidzin, Astragaloside IV respectively as alkaloids, flavonoids,
The internal standard compound of saponins compound assay, accurately weighed internal standard compound, adds methanol to be prepared into palmatin hydrochloride 0.984 respectively
The single inner mark solution of mg/mL, 1.080 mg/mL of daidzin, 1.392 mg/mL of Astragaloside IV；It is molten that precision draws single internal standard
Hydrochloric 78.72 ng/mL of palmatine, 216.00 ng/mL of daidzin, Astragaloside IV 556.80 is made with methanol dilution in liquid
The mixing inner mark solution of ng/mL；
(3) chromatography and Mass Spectrometry Conditions:
Chromatographic condition: chromatographic column is ACQUITY UPLC HSS T3 column (150 mm × 2.1 mm, 1.8 μm), mobile phase A: B
For acetonitrile: 0.1% formic acid water or A:B are 0.1% formic acid acetonitrile: 0.1% formic acid water, preferably 0.1% formic acid acetonitrile: 0.1% formic acid
Water, gradient elution program are 0~1.5 min, 17% A；1.5~3 min, 17 → 19% A；3~7 min, 19 → 33% A；7~
12 min, 33 → 98% A；12~14 min, 98 → 17% A；14~19 min, 17% A.Flow velocity is 0.3 mL/min；Column temperature
It is 40 DEG C；Sample volume is 3 μ L.
Mass Spectrometry Conditions: ion source: the source HESI；Positive and negative ion switches while scanning；Operating mode: Full-MS；Mass spectrum ginseng
Number: sheath gas: 35 arb；Secondary air speed: 10 arb；Spray voltage: 3.2 KV(+), 2.5 KV(-)；Capillary temperature:
320 ℃；Ion source temperature: 300 DEG C；Resolution ratio 70 000；Scanning range m/z 150-1500 Da, the mass spectrum of 12 kinds of ingredients
Parameter is shown in Table 1.
The mass spectrometry parameters of 1 12 kinds of ingredients of table
(4) processing of plasma sample: primarily looked in plasma sample treatment process precipitation of protein (methanol and acetonitrile) and
Liquid-liquid extraction method (ethyl acetate) is to the rate of recovery of 9 kinds of ingredients and the influence of matrix effect.Methanol extraction egg is used as the result is shown
Kaempferol -3- when whiteOThe rate of recovery of rutinoside is lower, and making to be extracted with ethyl acetate leads to magnoflorine, Wei Caining, this skin
Promise element, swertisin and 6'''The rate of recovery of asafoetide acyl spinosin is extremely low, finally selects have higher recovery to ingredient to be measured
Reliable solvent with the acetonitrile of low matrix effect as protein precipitation.Next compares different proportion acetonitrile (3 times, 4 times, 5
Times) sedimentation effect, the results show that the extraction recovery highest of the acetonitrile precipitation albumen Wei Caining using 3 times of volumes, other at
The rate of recovery divided is suitable, therefore selects the acetonitrile precipitation plasma protein of 3 times of volumes.
Precision draws 100 μ L of blank plasma, and 10 μ L internal standards are added, and be vortexed 30 s, then is separately added into 10 μ L working solutions
Be vortexed 30 s, and 300 μ L acetonitriles, 5 min of vortex are then added, is centrifuged (13000 rpm, 10 min, 4 DEG C), takes 350 μ of supernatant
L is added in clean EP pipe, 1400 rpm traditional vacuums, 40 DEG C of air blow dryings, and residue adds 100 μ L initial liquid phases vortex 2
Min is centrifuged (13000 rpm, 5 min, 4 DEG C), and supernatant is taken to carry out UHPLC-MS analysis.
Embodiment 2: methodological study
1. specificity is tested: blank plasma, the blank plasma for fetching 6 different rats of source add working solution, plasma containing drug equal
According to the condition processing and analysis of embodiment 1.Compare the chromatogram of three, observation is empty in ingredient to be measured and interior target appearance position
White blood plasma is with the presence or absence of interference.As a result as shown in Figure 1, ingredient to be measured and interior target retention time are respectively as follows: coclaurine 3.23
Min, 4.12 min of magnoflorine, dimension adopt peaceful 2.85 min, 6.13 min of spinosin, 6.87 min of swertisin, Kaempferol-
3-ORutinoside 7.37 min, 6'''Asafoetide acyl spinosin 8.14 min, 10.76 min of Saponin A, semen ziziphi spinosae
11.14 min of saponin(e B, 10.22 min of palmatin hydrochloride, 4.60 min of daidzin, 11.10 min of Astragaloside IV.It is to be measured at
Divide blank plasma corresponding with interior target appearance position not interfere with, shows that method specificity is good.
2. the range of linearity and lower limit of quantitation: using the ratio of Component peak area to be measured and internal standard peak area as ordinate (y),
The concentration for the ingredient reference substance to be measured being added in blood plasma is as abscissa (x), using 1/c2Weighted least-squares method carries out linear
It returns, finds out regression curve equation.In a few days complete the measurement of 3 standard curves.Lower limit of quantitation refer to signal-to-noise ratio (S/N) be greater than or
Equal to 10.The standard curves of 9 kinds of ingredients to be measured, related coefficient, the range of linearity and LLOQ the results are shown in Table 2, to be measured as the result is shown
Ingredient linear relationship in respective concentration range is good, related coefficientr 20.980~0.999.
The linearity and range of 29 kinds of ingredients of table,r 2 And LLOQ
3. precision and accuracy test: preci-sion and accuracy is measured using the QC sample of 4 concentration.Withinday precision
Using sample continuous sample introduction 5 times of same analysis batch, day to day precision analyzes sample continuous sample introduction 3 days criticized using 3, each
Analysis day continuous sample introduction 5 times calculates precision RSD % and accuracy RE%.9 kinds of ingredients to be measured in a few days, day to day precision and standard
Exactness the results are shown in Table 3, and withinday precision RSD% < 19.96%, accuracy % is -15.27%~17.31%；Day to day precision RSD%
< 19.62%, accuracy % is -14.33%~17.92%.Data show preci-sion and accuracy in critical field, as a result table
Bright this method has good preci-sion and accuracy, can be used for the analysis of biological sample.
39 kinds of ingredients of table in a few days, day to day precision and accuracy
4. matrix effect and extraction recovery are tested: matrix effect and extraction recovery are surveyed using the QC sample of 4 concentration
It is fixed.Matrix effect: plasma sample (n=5) are handled according to " processing of the plasma sample " method of embodiment 1, calculate separately ingredient to be measured
Value with internal standard peak area is A.100 μ L blank solvents (acetonitrile) are separately taken to replace blank plasma, other operations and 1 " blood of embodiment
The processing of slurry samples " processing method is consistent (n=5), and the value for calculating separately ingredient to be measured and internal standard peak area is B.Matrix effect=
A/B*100%, and calculate RSD value.Extraction recovery: blank plasma acetonitrile precipitation albumen post-processing makees solution by embodiment 1
" processing of plasma sample " method handles (n=5), and the value for calculating ingredient to be measured and internal standard peak area is C.Extraction recovery=A/
C*100%, and calculate RSD value.The extraction recovery range of 9 kinds of ingredients to be measured is 80.75%~113.75% in QC sample, matrix
Effect range be 77.67%~114.10%, RSD% < 15%, internal standard palmatin hydrochloride, daidzin, Astragaloside IV extraction recovery
Respectively 91.92%, 97.98%, 96.76%, matrix effect are respectively 108.93%, 101.38%, 98.49%, and the above results show
The extraction recovery of the sampling method of this experiment is good, and influences without significant matrix effect, the results are shown in Table 4.
The matrix effect and extraction recovery of 49 kinds of ingredients of table
5. stability test: carrying out study on the stability, including 25 DEG C of sample room temperature 4 h of placement of analysis to the QC sample of 4 concentration
Short-term stability, -20 DEG C place 30 days long-time stability and 3 Frozen-thawed cycleds after stability (- 20 DEG C~20 DEG C), with
And sample treatment is placed on the stability of 12 h in autosampler.Calculate precision RSD % and accuracy RE%.In different condition
4 are used under (25 DEG C of sample room temperature 4 h of placement, -20 DEG C place 30 days, 3 Frozen-thawed cycleds, place 12 h in autosampler)
Concentration QC sample studies the stability of 9 kinds of ingredients to be measured, the results are shown in Table 5.The result shows that all analytes are in entire analytic process
In it is stable, RE% is in -15.13%~19.78%, RSD% 0.38%~16.23%.
The stability of 59 kinds of ingredients of table
Embodiment 3: after the oral semen ziziphi spinosae water extract of normal and PCPA insomnia rat model in rat plasma 9 kinds of ingredients pharmacokinetics
Measurement
(1) preparation of semen ziziphi spinosae water extract: taking about 500 g of semen ziziphi spinosae medicine materical crude slice, crushes (60% crosses No. 1 pharmacopeia sieve), accurately weighed,
Add 10 times of amount water, impregnate 30 min, be heated to boiling, 2 h of refluxing extraction, filter, filter residue adds 8 times of amount water, heating and refluxing extraction 2 again
H, filtration, merging filtrate；It is concentrated under reduced pressure into 0.5 g/mL(crude drug meter), freeze-drying, the used time is dissolved with water.
(2) zoopery and the acquisition of plasma sample: healthy male SD rat, 280 ± 20 g of weight tie up tonneau by Beijing
Magnificent experimental animal Technology Co., Ltd. provides.Rat is randomly divided into 2 groups, normal group and model group, every group 6.With PCPA system
Standby insomnia model.400 mg/kg of PCPA solution is injected intraperitoneally in model group rats, continuous three days, after the last administration, observes rat daytime
Whether circadian rhythm disappears, the modeling success if disappearing.12 h of fasting before normal group and model group rats are administered, free water, mouth
Take 40 g/Kg(crude drug meter of semen ziziphi spinosae water extract).After administration in 0.083,0.167,0.333,0.5,0.75,1,2,4,6,
10,12,24 h retroorbital venous clumps take 300 μ L of blood, are placed in the centrifuge tube containing 0.1% sodium heparin anticoagulant, stand 30
Min, is centrifuged (3500 rpm, 10 min, 4 DEG C), and separated plasma sets -20 DEG C of preservations, for use.
(3) sample-pretreating method: precision draws 100 μ L of rat plasma, with plasma sample described in embodiment 1 processing
Method is handled.
(4) normal and PCPA insomnia rat model takes orally give semen ziziphi spinosae water extract after 9 kinds points of assay in blood plasma:
The plasma containing drug sample handled well is subjected to UHPLC-Q Exactive Orbitrap HR/MS analysis, condition is the same as implementation
Example 1 calculates plasma sample concentration according to standard curve.After normal group and model group rats take orally semen ziziphi spinosae water extract, 9 in blood plasma
Mean drug concentration-time graph of kind ingredient is shown in Fig. 2；Using DAS 3.2.8 data processing software, with non-room formula modelling into
Row pharmacokinetic parameter calculates, and the results are shown in Table 6.
The pharmacokinetic parameters of 9 kinds of ingredients after 6 normal groups of table (C) and model group (M) Oral Administration in Rats semen ziziphi spinosae water extract
Note: ND: blood concentration is lower than LLOQ.
As shown in Fig. 2, normal rat takes orally after 6 h of semen ziziphi spinosae water extract coclaurine, magnoflorine and Wei Caining in blood plasma
Blood concentration be lower than LLOQ, the blood concentration of swertisin is lower than LLOQ, Kaempferol -3- when 24 hORutinoside is being administered
Blood concentration is below LLOQ in 24 h；Rat model takes orally after 6 h of semen ziziphi spinosae water extract magnoflorine and Wei Caining in blood plasma
Blood concentration be lower than LLOQ.
C in semen ziziphi spinosae water extract in the content ratio and blood plasma of 9 kinds of ingredients to be measuredmaxAnd AUC0-tIt is not quite identical, it says
It is different that bright heterogeneity enters intracorporal degree.Some researches show that 6'''Asafoetide acyl spinosin, spinosin, semen ziziphi spinosae soap
The peak time of glycosides A and B are relatively long, but 9 kinds of ingredients to be measured of the invention reach peak in half an hour, show in semen ziziphi spinosae water extract
Other at their absorption may be promoted.
As shown in table 6, after normal rat takes orally semen ziziphi spinosae water extract, the T of coclaurine and magnoflorinemaxRespectively 0.2 h
With 0.46 h, CmaxRespectively 4.66 ng/mL and 140.29 ng/mL, T1/2Respectively 2.87 h and 1.15 h, it is seen that two kinds of lifes
The absorption of alkaloids is very fast, but the release rate of magnoflorine is faster than coclaurine.Spinosin and 6'''Asafoetide acyl Si Pinuo
The T of elementmaxRespectively 0.28 h and 0.36 h, CmaxRespectively 106.54 ng/mL and 48.45 ng/mL, T1/2Respectively 4.74
H and 2.21 h.Compared with spinosin, 6'''Asafoetide acyl spinosin absorbs slowly, and internal exposed amount is low, eliminates fast.We push away
This is surveyed the result is that due to 6'''There are asafoetide acyl group in asafoetide acyl spinosin structure, makes its fat-soluble increase, be easier to be distributed
In tissue.It is worth noting that, Double-peak Phenomenon occurs in swertisin, this may be as caused by two reasons: 1,6'''Ah
Wei's acyl spinosin and spinosin in enteron aisle constantly hydrolysis at swertisin；2, swertisin may have occurred liver
Intestines circulation.In addition, Wei Caining is in the fast (T of the intracorporal absorption of normal ratmaxH) for 0.5, slow (T is eliminated1/2H) for 3.17.Herein
Under testing conditions, Kaempferol -3-OThe blood concentration of rutinoside is lower than LLOQ.The T of Saponin A and BmaxRespectively
3.46 h and 0.96 h, CmaxRespectively 57.81 ng/mL and 14.02 ng/mL, T1/2Respectively 2.44 h and 8.28 h.It can
See, the absorption of Spine Date Seed jujubosideB is faster than Saponin A, and eliminates extremely slow.
For composition of alkaloids, compared with normal rat, coclaurine after the oral semen ziziphi spinosae water extract of rat model
Cmax、AUC0-tAnd T1/2Increase, TmaxReduce, illustrates that rat model accelerates the absorption of coclaurine, elimination slows down (P < 0.05), body
Interior exposed amount significantly improves；The C of magnoflorinemaxAnd AUC0-tReduce, TmaxIt reduces, CL/F is obviously increased, and illustrates rat model pair
The absorption of magnoflorine and release rate are accelerated, but its internal exposed amount reduces.
For flavones carbon carbohydrate content, compared with normal rat, rat model is taken orally 6 after semen ziziphi spinosae water extract'''-
The T of asafoetide acyl spinosinmaxIt is substantially reduced (P < 0.05), illustrates that rat model absorbs quickening to it；T1/2Significantly improve (P <
0.05), illustrate that release rate slows down；AUC0-tIt reduces, illustrates that its internal exposed amount is reduced.It is higher for content in semen ziziphi spinosae
Spinosin, the C of rat modelmaxAnd AUC0-tIt increases, TmaxReduce, shows that it is absorbed and accelerate, internal exposed amount increases；CL/F
Reduce, illustrates that its supersession rate slows down.The C of rat model swertisinmax、AUC0-¥And T1/2Dramatically increase (P < 0.05), CL/F
It is obviously reduced, illustrates rat model to its influx and translocation, internal exposed amount dramatically increases, and release rate slows down.Rat model dimension
Adopt peaceful CmaxIncrease, TmaxAnd T1/2Reduce, shows that rat model accelerates the absorption of Wei Caining and elimination.It can be seen that
Using apiolin as the 6 of parent nucleus'''Asafoetide acyl spinosin, spinosin and swertisin add in rat model body absorption
Fastly, and release rate slows down.Wherein the internal exposed amount of spinosin and swertisin increases.It and is all that the Wei Caining of carbon sugar exists
It equally shows to absorb the trend accelerated in rat model body.But its T1/2Shorten, eliminates and accelerate.Compared with normal rat, flavones
Oxygen sugar Kaempferol -3-ORutinoside exposed amount in rat model body obviously increases.
For saponin component Saponin A higher for content in semen ziziphi spinosae and B, compared with normal rat, acid
Jujube kernel saponin A is in the intracorporal T of rat model1/2Increase, TmaxIt is significantly reduced with CL/F, illustrates that rat model Saponin A is inhaled
It receives and accelerates (P < 0.05), elimination slows down (P < 0.05), and the trend that exposed amount is reduced in vivo.Spine Date Seed jujubosideB is big in model
The intracorporal AUC of mouse0-tIt is consistent substantially with CL/F with normal rat.However, TmaxIt shows reduction trend, illustrates up to peak more
Fastly.It can be seen that Saponin A and B accelerate in rat model body absorption；And Saponin A release rate is significant
It reduces, the release rate of Spine Date Seed jujubosideB is basically unchanged.
In the semen ziziphi spinosae water extract body that the present invention establishes while 9 kinds of active constituents quantitative approach can be applied to it is normal and
PCPA insomnic rats take orally the pharmacokinetic studies after semen ziziphi spinosae water extract.
Claims (7)
1. 9 kinds of method for quantitatively determining while enter blood component in a kind of semen ziziphi spinosae water extract, using UHPLC-Q Exactive
Orbitrap HR/MS method, be both 9 kinds of active constituents in quantitative determination semen ziziphi spinosae water extract: coclaurine, magnoflorine, dimension are adopted
Rather, spinosin, swertisin, Kaempferol -3-ORutinoside, 6'''Asafoetide acyl spinosin, Saponin A and acid
Jujube kernel saponin(e B；It is characterized by: specific step is as follows:
(1) preparation of single reference substance stock solution: UHPLC-Q Exactive is added in accurately weighed reference substance respectively
The chromatography initial liquid phase of Orbitrap HR/MS analysis, is prepared into 2.032 mg/mL of coclaurine, 1.130 mg/ of magnoflorine
The single reference substance stock solution of mL, addition methanol are prepared into dimension and adopt peaceful 2.184 mg/mL, 2.016 mg/mL of spinosin, work as medicine
1.972 mg/mL of flavine, Kaempferol -3-O2.058 mg/mL of rutinoside, 6'''2.012 mg/ of asafoetide acyl spinosin
The single reference substance stock solution of mL, 2.040 mg/mL of Saponin A, 2.064 mg/mL of Spine Date Seed jujubosideB；
The preparation of working solution: it is molten that mixing reference substance is made in the above-mentioned single reference substance stock solution addition methanol of accurate measurement respectively
Liquid, in which: 325.12 ng/mL of coclaurine, 18080 ng/mL of magnoflorine, Wei Caining, 2419.2 ng/mL of spinosin, when
94.66 ng/mL of medicine flavine, Kaempferol -3-O246.96 ng/mL of rutinoside, 6'''Asafoetide acyl spinosin 128.77
Ng/mL, 652.8 ng/mL of Saponin A, 412.8 ng/mL of Spine Date Seed jujubosideB；Then accurate to measure above-mentioned mixing pair
According to product solution, respectively plus methanol is diluted to the 4/5 of mixed reference substance solution concentration, 1/2,1/4,1/8,1/40,1/80 times step by step,
It shakes up to get the working solution of mass concentration gradient；
(2) preparation of inner mark solution: select palmatin hydrochloride, daidzin, Astragaloside IV respectively as alkaloids, flavonoids,
The internal standard compound of saponins compound assay, accurately weighed internal standard compound, adds methanol to be prepared into palmatin hydrochloride 0.984 respectively
The single inner mark solution of mg/mL, 1.080 mg/mL of daidzin, 1.392 mg/mL of Astragaloside IV；It is molten that precision draws single internal standard
Hydrochloric 78.72 ng/mL of palmatine, 216.00 ng/mL of daidzin, Astragaloside IV 556.80 is made with methanol dilution in liquid
The mixing inner mark solution of ng/mL；
(3) foundation of standard curve: precision draws 100 μ L of blank plasma, and 10 μ L internal standards are added, and be vortexed 30 s, then adds respectively
Enter 10 μ L hybrid working solution, 30 s of vortex, 300 μ L acetonitriles vortex 5 min, 13000 rpm, 4 DEG C of centrifugations 10 are then added
Min takes 350 μ L of supernatant to be added in clean EP pipe, and 1400 rpm traditional vacuums, 40 DEG C of air blow dryings, residue add 100
μ L initial liquid phase vortex 2 min, 13000 rpm, 4 DEG C of 5 min of centrifugation, take supernatant to carry out UHPLC-Q Exactive
Orbitrap HR/MS analysis；Using the ratio of Component peak area to be measured and internal standard peak area as ordinate (y), it is added in blood plasma
Ingredient reference substance to be measured concentration as abscissa (x), using 1/c2Weighted least-squares method carries out linear regression, establishes bent
Line；
(4) detection of sample:
(a) preparation of semen ziziphi spinosae water extract: 500 g of semen ziziphi spinosae medicine materical crude slice is crushed to 60% and crosses No. 1 pharmacopeia sieve, adds 10 times of amount water, soak
30 min are steeped, are heated to boiling, 2 h of refluxing extraction is filtered, and filter residue adds 8 times of amount water again, and 2 h of heating and refluxing extraction is filtered, and is merged
Filtrate；It is concentrated under reduced pressure into crude drug meter concentration as 0.5 g/mL, freeze-drying, the used time is dissolved with water；
(b) acquisition of plasma sample: Oral Administration in Rats semen ziziphi spinosae water extract with crude drug meter concentration be 40 g/Kg, after administration in
To rat 300 μ L of blood sampling during 0.083-24 h, it is placed in the centrifuge tube containing 0.1% sodium heparin anticoagulant, stands 30
Min, 4 DEG C, 3500 rpm, 10 min of centrifugation, separated plasma set -20 DEG C of preservations, for use；
(c) detection of sample: precision draws 100 μ L of rat plasma, is handled using the processing method of blank plasma samples,
For the solvent used in plasma sample processing for acetonitrile, dosage is 3 times of plasma sample；The plasma containing drug sample handled well is carried out
UHPLC-Q Exactive Orbitrap HR/MS analysis, the concentration of 9 kinds of ingredients in plasma sample is calculated according to standard curve.
2. 9 kinds of method for quantitatively determining while enter blood component in a kind of semen ziziphi spinosae water extract according to claim 1, special
Sign is: the chromatographic condition of the UHPLC-Q Exactive Orbitrap HR/MS analysis: chromatographic column is ACQUITY UPLC
HSS T3 column, the mm of 150 mm × 2.1,1.8 μm, mobile phase A: B is acetonitrile: 0.1% formic acid water or A:B are 0.1% formic acid second
Nitrile: 0.1% formic acid water, gradient elution program are 0~1.5 min, 17% A；1.5~3 min, 17 → 19% A；3~7 min, 19
→33% A；7~12 min, 33 → 98% A；12~14 min, 98 → 17% A；14~19 min, 17% A；Flow velocity is 0.3
mL/min；Column temperature is 40 DEG C；Sample volume is 3 μ L；
Mass Spectrometry Conditions: ion source: the source HESI；Positive and negative ion switches while scanning；Operating mode: Full-MS；Mass spectrometry parameters: sheath
Gas velocity: 35 arb；Secondary air speed: 10 arb；Spray voltage: 3.2 KV(+), 2.5 KV(-)；Capillary temperature: 320
℃；Ion source temperature: 300 DEG C；Resolution ratio 70 000；Scanning range m/z 150-1500 Da.
3. 9 kinds of method for quantitatively determining while enter blood component in a kind of semen ziziphi spinosae water extract according to claim 1, special
Sign is: the chromatography initial liquid phase of the analysis of UHPLC-Q Exactive Orbitrap HR/MS described in step (1) is 17%
0.1% formic acid acetonitrile -83% 0.1% formic acid water.
4. 9 kinds of method for quantitatively determining while enter blood component in a kind of semen ziziphi spinosae water extract according to claim 1, special
Sign is: in the step (b) respectively rat administration after 0.083,0.167,0.333,0.5,0.75,1,2,4,6,10,12,
Blood was collected to rat by 24 h.
5. 9 kinds of method for quantitatively determining while enter blood component in a kind of semen ziziphi spinosae water extract according to claim 1, special
Sign is: the solvent used in the blank plasma samples processing is any one in methanol, acetonitrile or ethyl acetate, dosage
It is 3-5 times of plasma sample.
6. 9 kinds of method for quantitatively determining while enter blood component in a kind of semen ziziphi spinosae water extract according to claim 4, special
Sign is: for the solvent used in the blank plasma samples processing for acetonitrile, dosage is 3 times of plasma sample.
7. 9 kinds of method for quantitatively determining while enter blood component in a kind of semen ziziphi spinosae water extract according to claim 2, special
Sign is: mobile phase A in chromatographic condition: B is 0.1% formic acid acetonitrile: 0.1% formic acid water.
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| CN110715994B (en) * | 2019-09-30 | 2021-12-17 | 山西大学 | Method for analyzing difference chemical components of spina date seed and spina date seed by using UHPLC-Q-Orbitrap MS |
| CN115684371A (en) * | 2021-07-26 | 2023-02-03 | 泰州医药城国科化物生物医药科技有限公司 | Rapid liquid chromatography-mass spectrometry quantification method for saponin in spina date seed aqueous extract |
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| CN114487181B (en) * | 2022-01-20 | 2024-03-19 | 广西壮族自治区食品药品检验所 | Method for measuring content of spine date seed saponin A and saponin B in Tianwang heart tonifying preparation |
| CN114948980A (en) * | 2022-06-09 | 2022-08-30 | 山西大学 | Pharmaceutical composition for preventing and/or treating anxiety insomnia and application thereof |
| CN114948980B (en) * | 2022-06-09 | 2024-03-12 | 山西大学 | Pharmaceutical composition for preventing and/or treating anxiety insomnia and application thereof |
| CN115372520A (en) * | 2022-09-20 | 2022-11-22 | 贵州医科大学 | Blood-entering component and component distribution determination method of litsea cubeba oil |
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