CN103983732A - Determination method for two components in plasma of Relinqing granules - Google Patents
Determination method for two components in plasma of Relinqing granules Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 85
- 239000008187 granular material Substances 0.000 title abstract 4
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 claims abstract description 70
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims abstract description 70
- 229940074391 gallic acid Drugs 0.000 claims abstract description 35
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- 239000013558 reference substance Substances 0.000 claims description 58
- 239000002253 acid Substances 0.000 claims description 56
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 55
- 235000019253 formic acid Nutrition 0.000 claims description 55
- 239000000243 solution Substances 0.000 claims description 52
- 239000002245 particle Substances 0.000 claims description 48
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- 238000012360 testing method Methods 0.000 claims description 31
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
- 239000007921 spray Substances 0.000 claims description 27
- 239000007864 aqueous solution Substances 0.000 claims description 26
- YWJXCIXBAKGUKZ-HJJNZUOJSA-N Bergenin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@H]2C3=C(O)C(OC)=C(O)C=C3C(=O)O[C@@H]21 YWJXCIXBAKGUKZ-HJJNZUOJSA-N 0.000 claims description 20
- XULPLJSODQQHPH-UHFFFAOYSA-N Bergenin Natural products OCC1OC2C(OC(=O)c3cc(O)c(CO)c(O)c23)C(O)C1O XULPLJSODQQHPH-UHFFFAOYSA-N 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 18
- 229920000669 heparin Polymers 0.000 claims description 18
- 239000012452 mother liquor Substances 0.000 claims description 18
- 230000006920 protein precipitation Effects 0.000 claims description 17
- 239000007789 gas Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- 238000000889 atomisation Methods 0.000 claims description 15
- 238000001802 infusion Methods 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 15
- 238000007710 freezing Methods 0.000 claims description 14
- 230000002496 gastric effect Effects 0.000 claims description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- 238000001556 precipitation Methods 0.000 claims description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims description 13
- 238000012544 monitoring process Methods 0.000 claims description 13
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- 239000003480 eluent Substances 0.000 claims description 12
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 10
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 8
- 239000012503 blood component Substances 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 6
- 239000012490 blank solution Substances 0.000 claims description 6
- 229960002897 heparin Drugs 0.000 claims description 6
- 238000003916 acid precipitation Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 4
- 238000001819 mass spectrum Methods 0.000 claims description 4
- 230000037396 body weight Effects 0.000 claims description 2
- 238000001647 drug administration Methods 0.000 claims description 2
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- 238000001514 detection method Methods 0.000 abstract description 5
- 239000012071 phase Substances 0.000 description 50
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- 241000700159 Rattus Species 0.000 description 23
- 150000002500 ions Chemical class 0.000 description 23
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 description 15
- 238000000605 extraction Methods 0.000 description 13
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- 239000000126 substance Substances 0.000 description 5
- 241000764065 Persicaria capitata Species 0.000 description 3
- KWGRBVOPPLSCSI-WPRPVWTQSA-N (-)-ephedrine Chemical compound CN[C@@H](C)[C@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WPRPVWTQSA-N 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
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- DLYUQMMRRRQYAE-UHFFFAOYSA-N tetraphosphorus decaoxide Chemical compound O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 2
- 208000004880 Polyuria Diseases 0.000 description 1
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- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for determining two components in plasma of Relinqing granules and for content determination in the plasma thereof. The content determination method adopts a high performance liquid chromatography-mass spectrometry detection method, and content determination is implemented on components in plasma, i.e., gallic acid and protocatechuic acid, in the Relinqing granules through the established detection method. The content determination method disclosed by the invention is good in precision degree, good in reproducibility, good in stability and accurate in determination result, and is capable of effectively controlling mass of the Relinqing granules.
Description
Technical field
The invention belongs to Chinese patent drug internal metabolism studying technological domain, be specifically related to a kind of heat drench in clear particle two kinds enter blood component determine and blood plasma in content assaying method.
Technical background
It is to take the single preparations of ephedrine that seedling medicine polygonum capitatum makes as raw material that heat is drenched clear particle, records in < < Chinese Pharmacopoeia > > 2010 editions.Tool is clearing heat and detoxicating, the effect of inducing diuresis for treating strangurtia.For damp-heat accumulation, dark urine, the disease of odynuria, urinary tract infections, pyelonephritis is shown in above-mentioned patient.Urinary system infection contamination is had to definite curative effect." heat is drenched clear particle " is national Chinese medicine protection kind, national social security medicine; and be unique seedling medicine that enters 2010 editions < < Chinese Pharmacopoeia > >, obtain the independent pricing right of National Development and Reform Committee.
According to bibliographical information, the research of at present drenching clear particle for heat mainly concentrates on the aspects such as chemical constitution study, quality controling research and pharmacology activity research.But the effective substance that heat is drenched clear particle performance effect is not also too clear, the detection means of effective constituent in blood lacks.For the oral heat of further further investigation, drench after clear particle, the situation of change of effective constituent in blood is necessary to develop a kind of detection technique accurately and reliably very much, to provide scientific basis for illustrating the effective substance of the clear particle performance of heat pouring effect.The present invention completes just under this background.
Summary of the invention
The object of the invention is to: provide a kind of heat to drench the content assaying method in blood that clear particle enters blood component.The present invention enters blood chemistry composition gallic acid after clear particle is taken and the content of protocatechuic acid in blood is measured by heat is drenched, make up the deficiency that its existing detection technique lacks, for illustrating effective substance that heat the drenches clear particle performance effect behavior base in blood, provide science guarantee.
The present invention is achieved in that
Provide a kind of heat to drench two kinds of assay methods that enter blood component of clear particle, it comprises the following steps:
(1) preparation of test sample:
(a) heat-obtaining drenches clear particle 0.5～4g, adds 5～10 times of water-soluble solutions, gives rat (body weight 230-270g) heat drench clear particle aqueous solution 10ml/kg by gastric infusion mode;
(b), respectively after administration 30～120min, to the rat 0.3～0.5ml that takes a blood sample, by after the centrifugal 5～30min of blood sample 3000～5000r/min rotating speed with liquaemin anti-freezing, get upper plasma and be transferred in new blank EP pipe;
(c) add interior mark Bergenin mother liquor 10 μ l, then by blood plasma and precipitation reagent volume ratio, be 1:4～8 protein precipitation, settling time 2～5min, vortex mixes after 30～60s, under 0～10 ℃ of constant temperature, take the centrifugal 5～15min of 10000～12000r/min (described precipitation reagent be acetonitrile-methyl alcohol (3:2) mixed liquor and add 1%～4% acid, described acid is selected from formic acid, acetic acid or its combination)
(d) upper strata liquid is transferred in new blank EP pipe, in 30～50 ℃ of nitrogen streams, dry up, residue dissolves with 100 μ l mobile phases, and vortex mixes after 30～60s, under 0～10 ℃ of constant temperature, with the centrifugal 5～15min of 10000～12000r/min, get supernatant and be need testing solution;
(e) by step (b) to the method for step (d), the blank plasma of drug administration is not processed, obtained blank solution;
(f) preparation of reference substance solution: precision takes gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg respectively, add separately pure methanol constant volume to 10ml, obtain three kinds of mother liquors, get respectively three kinds of mother liquors appropriate to same measuring bottle, add methyl alcohol and be diluted to desired concn, be reference substance solution;
(2) LC-MS/MS analyzes chromatographic condition and testing conditions:
Chromatographic column is reverse-phase chromatographic column, acid acetonitrile or acidic methanol: acidulous water=0～20:80～100 (volume ratio) is mobile phase, flow velocity is 0.1～0.5mL/min, column temperature is 20～40 ℃, ionization mode is electron spray condition, negative mode monitoring, spray voltage is 2000～3000V, atomization temperature is 300～500 ℃, sheath atmospheric pressure is 30～50mTorr, assisted gas pressure is 5～15mTorr, capillary temperature is 200～400 ℃ (in described acid acetonitrile or acidic methanol or acidulous water, the concentration of acid is 0.05%～0.5%, described acid is selected from formic acid, acetic acid or its combination),
(3) determination method: precision is drawn reference substance solution, blank solution and need testing solution 5～20 μ L respectively, injects HPLC-MS instrument, measures, and obtains.
The method according to this invention, wherein, in step (a), drenches clear particle aqueous solution 2～4ml by gastric infusion mode to rat (230～270g) heat.
The method according to this invention, wherein in step (b), respectively at 30min, 60min, after 120min, rat is taken a blood sample.
The method according to this invention, wherein in step (b), by blood in the centrifugal 10min of low speed centrifuge 4000r/min.
The method according to this invention, in step (c), is wherein 2% acid acetonitrile-methyl alcohol (3:2) 1:4 protein precipitation 5min by volume by acidity.
The method according to this invention, wherein, in step (c), after protein precipitation, after the even 30s of vortex, 4 ℃ of constant temperature are centrifugal, rotating speed 12000r/min, centrifugal 10min.
The method according to this invention, wherein, in step (c), is used formic acid: acetic acid=2:1 volume ratio acid mixture carrys out acid precipitation reagent.
The method according to this invention, wherein two kinds to enter blood component be gallic acid and protocatechuic acid.
The method according to this invention, wherein, in step (d), dries up in Nitrogen evaporator supernatant liquor in 40 ℃.
The method according to this invention, wherein, in step (d), residue dissolves with 100 μ l mobile phases, and vortex mixes 12000r/min after 60s, and 4 ℃ of constant temperature, get 10 μ l sample introductions after centrifugal 10min.
The method according to this invention, wherein, in step (2), chromatographic column is C18 chromatographic column.
The method according to this invention, wherein, in step (2), in described acid acetonitrile or acidic methanol or acidulous water, the concentration of acid is 0.1%.
The method according to this invention, wherein, in step (2), flow velocity is 0.2ml/min, column temperature is 30 ℃.
The method according to this invention, wherein, in step (2), ionization mode is electron spray condition, negative mode monitoring, spray voltage is 2500V, and atomization temperature is 350 ℃, and sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, capillary temperature is 300 ℃.
The method according to this invention, wherein in step (2), is used gradient elution, being changed to of eluent: during beginning, and acid acetonitrile mutually 2%, acidic aqueous solution mutually 98%, stablizes 3min; During then to 10min, acid acetonitrile mutually linearity is increased to 10%, acidic aqueous solution phase 90%; During 10.1min, acid acetonitrile changes 2% mutually into, and acidic aqueous solution phase 98%, stablizes 7min.
The method according to this invention, wherein, in step (3), accurate reference substance solution, blank solution and the need testing solution 5 μ L of drawing, inject HPLC-MS instrument respectively.
The method according to this invention, wherein in step (2), Mass Spectrometer Method is multiple-reaction monitoring pattern, reactive ion to and form the required collision energy of stabistor ion and be respectively: gallic acid 169.181 → 125.268, bucking voltage (T μ be Lens Offset) 71V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV; Protocatechuic acid 152.918 → 109.244, bucking voltage (T μ be Lens Offset) 75V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV; Interior mark Bergenin 326.922 → 192.167, bucking voltage (T μ be Lens Offset) 100V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 22eV.
The method according to this invention, it is characterized in that: heat-obtaining drenches clear particle 0.5～4g, add 5～10 times of water-soluble solutions, by gastric infusion mode, to rat (230-270g) heat, drench clear particle aqueous solution 10ml/kg, in 30～120min to the rat 0.3～0.5ml that takes a blood sample, be placed in liquaemin anti-freezing test tube, after the centrifugal 5～30min of blood sample 3000～5000r/min rotating speed, getting upper plasma is transferred in new blank EP pipe, add interior mark Bergenin 10 μ l, then press 1%～4% acid acetonitrile-methyl alcohol (3:2) protein precipitation for 1:4～1:8 volume ratio, settling time 2～5min, vortex mixes 10000～12000r/min after 30～60s, 0～10 ℃ of centrifugal 5～15min of constant temperature, upper strata liquid is transferred in new blank EP pipe and dries up in 30～50 ℃ of nitrogen streams, residue dissolves with 100 μ l mobile phases, vortex mixes 10000～12000r/min after 30～60s, 0～10 ℃ of constant temperature, after centrifugal 5～15min, get 5～20 μ l sample introductions, blank plasma is pressed same method and is processed, adopt liquid chromatograph-mass spectrometer to carry out LC-MS/MS analysis by following condition:
Chromatographic condition and testing conditions: chromatographic column is reverse-phase chromatographic column, acid (formic acid or acetic acid 0.05%～0.5%) acetonitrile or methyl alcohol: acid (formic acid or acetic acid 0.05%～0.5%) water=0～20:80～100 (volume ratio) is mobile phase, gradient elution, flow velocity is 0.1～0.5mL/min, column temperature is 20～40 ℃, ionization mode is electron spray condition, negative mode monitoring, spray voltage is 2000～3000V, atomization temperature is 300～500 ℃, sheath atmospheric pressure is 30～50mTorr, and assisted gas pressure is 5～15mTorr, and capillary temperature is 200～400 ℃;
The preparation of reference substance solution: get gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg, accurately weighed, add methanol constant volume to 10ml, obtain mother liquor, working fluid is that mother liquor adds methyl alcohol and is diluted to desired concn.
According to the present invention, described reverse-phase chromatographic column is C18 chromatographic column.
The method according to this invention, is characterized in that: plasma sample operates according to the following steps:
Heat-obtaining drenches clear particle 0.5～4g, add 5～10 times of water-soluble solutions, by gastric infusion mode, to rat (230-270g) heat, drench clear particle aqueous solution 10ml/kg, respectively at 30～120min, afterwards to the rat 0.3～0.5ml that takes a blood sample, by after the centrifugal 10min of blood sample 4000r/min rotating speed with liquaemin anti-freezing, getting upper plasma is transferred in new blank EP pipe, add interior mark Bergenin 10 μ l, then press 2% acid acetonitrile-methyl alcohol (3:2) protein precipitation for 1:4 volume ratio, settling time 5min, vortex mixes the centrifugal 5min of rear 12000r/min, upper strata liquid is transferred in new blank EP pipe and dries up in 40 ℃ of nitrogen streams, residue dissolves with 100 μ l mobile phases, 4 ℃ of constant temperature, after the centrifugal 10min of 12000r/min, get 10 μ l sample introductions, blank plasma is pressed same method and is processed.
The method according to this invention, it is characterized in that: chromatographic column specification is 2.1 * 150mm, packing material size is 1.7 μ m, take formic acid 0.1% acetonitrile A: formic acid 0.1% water B is mobile phase, press following condition gradient elution: 0～3min2%A, 3.0～3.1min2%A-10%A, 3.1～10.0min10%A, 10.0～10.1min10%-2%A, 10.1～17.0min2%A.
The method according to this invention, is characterized in that: mass spectrum spray voltage is 2500V, and atomization temperature is 350 ℃, and sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, and capillary temperature is 350 ℃.
The method according to this invention, is characterized in that: Mass Spectrometer Method is multiple-reaction monitoring pattern, and reactive ion is to being respectively: gallic acid 169.181 → 125.268, protocatechuic acid 152.918 → 109.244, interior mark 326.922 → 192.167.
The method according to this invention, it preferably includes following steps:
Heat-obtaining drenches 5～10 times of water-soluble solutions for clear particle 0.5～4g, be made into aqueous solution, by gastric infusion mode, to rat (230～270g) heat, drench clear particle aqueous solution 2～4ml, respectively at 30min, 60min, after 120min, rat is taken a blood sample, heparin tube is used liquaemin anti-freezing in advance, by blood after the centrifugal 10min of low speed centrifuge 4000r/min, getting upper plasma is transferred in new blank EP pipe, by acidity, be 2% acid acetonitrile-methyl alcohol (3:2) 1:4 protein precipitation 5min by volume, after the even 30s of vortex, 4 ℃ of constant temperature are centrifugal, rotating speed 12000r/min, centrifugal 10min, supernatant liquor is dried up in 40 ℃ in Nitrogen evaporator, residue dissolves with 100 μ l mobile phases, vortex mixes 12000r/min after 60s, 4 ℃ of constant temperature, after centrifugal 10min, get 10 μ l sample introductions, blank plasma is pressed same method and is processed, adopt liquid chromatograph-mass spectrometer to carry out LC-MS/MS by following condition:
Chromatographic condition and testing conditions: chromatographic column is reverse-phase chromatographic column, acid (formic acid 0.1%) acetonitrile: acid (formic acid 0.1%) water=0～20:80～100 (volume ratio) are mobile phase, gradient elution, flow velocity is 0.2ml/min, column temperature is 30 ℃, ionization mode is electron spray condition, negative mode monitoring, and spray voltage is 2500V, atomization temperature is 350 ℃, sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, and capillary temperature is 300 ℃;
The preparation of reference substance solution: get gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg, accurately weighed, add pure methanol constant volume to 10ml, obtain mother liquor, working fluid is that mother liquor adds methyl alcohol and is diluted to desired concn;
Determination method: precision is drawn reference substance solution and need testing solution 5 μ L respectively, injects HPLC-MS instrument, measures, and obtains.
In aforementioned eluent gradient elution process, being changed to of eluent: during beginning, formic acid acetonitrile mutually 2%, aqueous formic acid mutually 98%, stablizes 3min; During 10min, formic acid acetonitrile phase 10%, during aqueous formic acid phase 90%, the 10.1min, formic acid acetonitrile phase 2%, aqueous formic acid phase 98%, stablizes 7min.
Aforementioned Mass Spectrometer Method is multiple-reaction monitoring pattern, reactive ion to and form the required collision energy of stabistor ion and be respectively: gallic acid 169.181 → 125.268, bucking voltage (T μ be Lens Offset) 71V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, protocatechuic acid 152.918 → 109.244, bucking voltage (T μ be Lens Offset) 75V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, interior mark 326.922 → 192.167, bucking voltage (T μ be Lens Offset) 100V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 22eV.
Being below the inventor drenches in clear particle, to enter blood chemistry component content and measure the process of studying to heat:
One, the clear particle of oral heat pouring enters determining of blood chemistry composition
The document that the inventor is drenched clear particle and bulk drug thereof for relevant heat has carried out a large amount of research, and chemical constitution study early stage polygonum capitatum being launched according to inventor place seminar is found, gallic acid and protocatechuic acid are the bioactive ingredients in polygonum capitatum, and gallic acid and the protocatechuic acid therefore chosen in the clear particle of heat pouring are that index has carried out entering blood test research.
Heat-obtaining drenches 5～10 times of water-soluble solutions for clear particle 0.5～4g, be made into aqueous solution, by gastric infusion mode, to rat (230～270g) heat, drench clear particle aqueous solution 2～4ml, respectively at 30min, 60min, after 120min, rat is taken a blood sample, heparin tube is used liquaemin anti-freezing in advance, by blood after the centrifugal 10min of low speed centrifuge 4000r/min, getting upper plasma is transferred in new blank EP pipe, by acidity, be 2% acid acetonitrile-methyl alcohol (3:2) 1:4 protein precipitation 5min by volume, after the even 30s of vortex, 4 ℃ of constant temperature are centrifugal, rotating speed 12000r/min, centrifugal 10min, supernatant liquor is dried up in 40 ℃ in Nitrogen evaporator, residue dissolves with 100 μ l mobile phases, vortex mixes 12000r/min after 60s, 4 ℃ of constant temperature, after centrifugal 10min, get 10 μ l sample introductions, blank plasma is pressed same method and is processed, adopt liquid chromatograph-mass spectrometer to carry out LC-MS/MS by following condition:
Chromatographic condition and testing conditions: chromatographic column is reverse-phase chromatographic column, acid (formic acid 0.1%) acetonitrile: acid (formic acid 0.1%) water=0～20:80～100 (volume ratio) are mobile phase, gradient elution, flow velocity is 0.2ml/min, column temperature is 30 ℃, ionization mode is electron spray condition, negative mode monitoring, and spray voltage is 2500V, atomization temperature is 350 ℃, sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, and capillary temperature is 300 ℃;
The preparation of reference substance solution: get gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg, accurately weighed, add methanol constant volume to 10ml, obtain mother liquor, working fluid is that mother liquor adds methyl alcohol and is diluted to desired concn.
Determination method: precision is drawn reference substance solution and need testing solution 5 μ L respectively, injects HPLC-MS instrument, measures, and obtains.
In aforementioned eluent gradient elution process, being changed to of eluent: during beginning, formic acid acetonitrile mutually 2%, aqueous formic acid mutually 98%, stablizes 3min; During 10min, formic acid acetonitrile phase 10%, during aqueous formic acid phase 90%, the 10.1min, formic acid acetonitrile phase 2%, aqueous formic acid phase 98%, stablizes 7min.
Aforementioned Mass Spectrometer Method is multiple-reaction monitoring pattern, reactive ion to and form the required collision energy of stabistor ion and be respectively: gallic acid 169.181 → 125.268, bucking voltage (T μ be Lens Offset) 71V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, protocatechuic acid 152.918 → 109.244, bucking voltage (T μ be Lens Offset) 75V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, interior mark 326.922 → 192.167, bucking voltage (T μ be Lens Offset) 100V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 22eV.
As Figure 1-4, as can be seen from Fig., gallic acid and protocatechuic acid drench and to enter blood chemistry composition in clear particle for heat measurement result.
Two, the foundation of the content assaying method of gallic acid and protocatechuic acid in the clear particle rat plasma of oral heat pouring
1. instrument and reagent
TSQ Q μ ant μ m Ultra Performance Liquid Chromatography-GC-MS (Μ PLC-MS/MS), comprise: triple level Four bar mass analyzers, ESI ion gun, Xcalib μ r workstation, liquid phase part is Thermo Accela Μ PLC, comprise: Accela PDA detecting device, Accela automatic sampler, Accela1250 infusion pump (U.S. Thermo Fisher Scientific Inc.), 100,000/electronic balance (plum Teller-Tuo benefit instrument Shanghai company limited), KQ5200E type Ultrasonic Cleaning (Kunming ultrasonic instrument company limited).
Acetonitrile (chromatographically pure, the U.S. " world " reagent company), methyl alcohol (chromatographically pure, the U.S. " world " reagent company); Water (redistilled water, before use preparation); Formic acid (chromatographically pure, U.S. Roe Scientific company), acetic acid (chromatographically pure, U.S. Roe Scientific company);
Gallic acid, protocatechuic acid and Bergenin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute provides).
2. instrumentation condition
Liquid phase chromatogram condition: Phenomenex KinetexXB-C18 chromatographic column (2.1 * 150mm, 1.7 μ m), mobile phase A is acetonitrile (containing 0.1% formic acid), and B is water (containing 0.1% formic acid), gradient elution: 0～3min2%A, 3.0～3.1min2%A-10%A, 3.1～10.0min10%A, 10.0～10.1min10%-2%A, 10.1～17.0min2%A, flow velocity 0.2mL/min, sample size is 5 μ L
Mass spectrum condition: adopt ESI ion gun; Negative ion detects pattern; Scan mode is multiple-reaction monitoring mode (MRM); Be used for the monitoring ion of quantitative test in Table 1.Source parameters: sheath gas velocity: 40Arb, assisted gas flow velocity: 10Arb; Capillary temperature: 350 ℃; Capillary voltage: 30V; Atomization temperature is 350 ℃; Spray voltage: 2500V; Sweep frequency: 0.1s.
Table 1, with SRM pattern optimize GA, PA, interior mark (IS) forms the required collision energy of stabistor ion
3. the selection of extraction conditions
Adopt respectively two kinds of extracting modes of Direct precipitation albumen and liquid-liquid extraction to extract, find that Direct precipitation protein extraction effect is better.Different precipitation agents, precipitation agent consumption and extraction time are examined or check, final definite extraction conditions is: with 2% acid acetonitrile-methyl alcohol (3:2) protein precipitation (1:4v/v), vortex 30s makes to mix, separation after albumen precipitation: 12000r/min is centrifugal, 4 ℃ of constant temperature, centrifugal 10min.The results are shown in Table 2-5
The impact of the different extracting modes of table 2 on GA and PA extraction efficiency
The impact on GA and PA extraction efficiency of the different precipitation agents of table 3 and precipitation agent consumption
The impact of the different precipitation agent acidity of table 4 on GA and PA extraction efficiency
The impact on GA and PA extraction efficiency of table 5 different extraction times
4, the investigation of linear relationship
Precision take through phosphorus pentoxide, be dried to constant weight gallic acid, protocatechuic acid and Bergenin 1.66mg in 10mL volumetric flask, add the reference substance storing solution that methyl alcohol is made into 0.166mg/mL.Be prepared into respectively gallic acid 30,60,300,600,1200,2400,3000ng/mL, protocatechuic acid 10, 20, 100, 200, 400, 600, the solution of each 7 variable concentrations of 1000ng/mL, accurate each the 5 μ L of this reference substance series solution that draw join in blank plasma respectively, by said extracted condition, operate, by above-mentioned chromatogram mass spectrum condition, measure, and to take the peak area of reference substance and the ratio of interior mark peak area be ordinate, concentration (ng/mL) is horizontal ordinate, with weighted least-squares method drawing standard curve, regression equation is respectively: gallic acid Y=0.006574X-0.047086, r2=0.9986, protocatechuic acid: Y=0.015679X-0.016621, r2=0.9977 result shows, gallic acid is at 30～3000ng/mL, protocatechuic acid is good in 10～1000ng/mL linear relationship.
5. precision test
Withinday precision, in standard curve range, is selected gallic acid, basic, normal, high 3 the concentration preparation quality Quality controls of protocatechuic acid, and each concentration sample introduction 6 times calculates withinday precision, and result meets the methodology requirement of Quantitative Study in biological sample.
Table 6 withinday precision
Continuous sample introduction 3 days, calculates day to day precision, and result meets the methodology requirement of Quantitative Study in biological sample, illustrates that the method has good precision.
Table 7 day to day precision
6, accuracy test
Get the quality-control sample of variable concentrations, carry out accuracy in computation with typical curve simultaneously.
Table 8 accuracy test result
7, recovery of extraction is investigated
Get respectively the standard plasma sample that contains different quality concentration in gallic acid, protocatechuic acid basic, normal, high 3, add inner mark solution, method by the inventive method step (1) it (c)-(d) is prepared need testing solution, record the peak area of reference substance, respectively at comparing with the measured peak area of inner mark solution sample introduction with the formulated same concentrations reference substance of mobile phase, calculate the recovery of gallic acid, protocatechuic acid in blood plasma.Result is as follows:
Table 9 extraction recovery (n=3) (%)
In addition, in supplementary test, the method of using with reference to above table 9, but what in step of the present invention (1) it (c), in precipitation reagent used, add is formic acid: during acetic acid=2:1 volume ratio acid mixture, in upper table 9, the extraction recovery of GA is all in 96～98% scopes, the extraction recovery of PA all, in 95～98% scopes, shows with acid mixture and comes acid precipitation reagent to have significantly better effect.But use the extraction recovery of formic acid/acetic acid=3/1, formic acid/acetic acid=4/1, formic acid/acetic acid=8/1, formic acid/acetic acid=1.5/1, formic acid/acetic acid=1/1, formic acid/acetic acid=0.5/1 o'clock GA and PA all lower than 88%.Therefore, in one embodiment of the invention, use formic acid: acetic acid=2:1 volume ratio acid mixture carrys out acid precipitation reagent.
8, study on the stability
Get blank plasma, add a certain amount of GA and PA standard solution, be mixed with certain density standard blood sample, during respectively at fresh configuration, room temperature places 4h, 8h, the analysis of 24h sample introduction after placing 4h, 8h and 24h and processing, in frozen process experiment, respectively at sampling after room temperature and-20 ℃ of freezing rear continuous three freeze thawing, by sample treatment, process sample introduction analysis after processing.Long-term stable experiment is-20 ℃ of freezing maintenances 7 days, then presses sample treatment and processes sample introduction analysis.Result is as follows:
The study on the stability of GA, PA (n=3) in table 10 plasma sample
The precision of content assaying method of the present invention is high, and favorable reproducibility is stablized, and the recovery is high, and measurement result is accurate, can be used for the assay that Oral Administration in Rats heat is drenched after clear particle gallic acid and protocatechuic acid in blood plasma.
In the present invention, term " acidity " is to point to the sour percentage concentration of adding in body liquid, also can be described as " concentration ", and for example " acidity is 2% " represents that the sour amount of adding in this body fluid is 2%.
Accompanying drawing explanation:
Fig. 1-4th, heat is drenched clear particle and is entered blood chemistry composition total ion current figure, wherein:
Fig. 1 is the total ion current figure (ordinate is relative absorbance log, and horizontal ordinate is retention time (min), lower same) of blank plasma;
Fig. 2 is the total ion current figure of 30 minutes plasma samples after rat administration;
Fig. 3 is the total ion current figure of 60 minutes plasma samples after rat administration;
Fig. 4 is the total ion current figure of 2 hours plasma samples after rat administration.
Embodiment:
Further illustrate by the following examples the present invention, but not as restriction of the present invention.In example below, if not otherwise specified, the acid of adding in mobile phase is formic acid, and concentration is 0.1%; The acid of adding in precipitation reagent is formic acid/acetic acid=2/1, and concentration is 2%.
embodiment 1:
Heat is drenched definite method that clear particle enters blood component: heat-obtaining drenches 5 times of water-soluble solutions for clear particle 0.5g, be made into the aqueous solution that concentration is 0.1g/ml, by gastric infusion mode, to rat (245g) heat, drench clear particle aqueous solution 2.5ml, after 30min, rat is taken a blood sample, heparin tube is used liquaemin anti-freezing in advance, by blood after the centrifugal 10min of low speed centrifuge 4000r/min, getting upper plasma is transferred in new blank EP pipe, by acidity, be 2% acid acetonitrile-methyl alcohol (3:2) 1:4 protein precipitation 5min by volume, after the even 30s of vortex, 4 ℃ of constant temperature are centrifugal, rotating speed 12000r/min, centrifugal 10min, supernatant liquor is dried up in 40 ℃ in Nitrogen evaporator, residue dissolves with 100 μ l mobile phases, vortex mixes 12000r/min after 60s, 4 ℃ of constant temperature, after centrifugal 10min, get 10 μ l sample introductions, blank plasma is pressed same method and is processed, adopt liquid chromatograph-mass spectrometer to carry out LC-MS/MS analysis by following condition:
Chromatographic condition and testing conditions: chromatographic column is anti-phase C18 chromatographic column, acid (formic acid 0.1%) acetonitrile: acid (formic acid 0.1%) water=0～20:80～100 (volume ratio) are mobile phase, gradient elution, flow velocity is 0.2ml/min, column temperature is 30 ℃, ionization mode is electron spray condition, negative mode monitoring, and spray voltage is 2500V, atomization temperature is 350 ℃, sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, and capillary temperature is 300 ℃;
The preparation of reference substance solution: get gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.2mg, accurately weighed, add pure methanol constant volume to 10ml, obtain mother liquor, working fluid is that mother liquor adds methyl alcohol and is diluted to desired concn.
Determination method: precision is drawn reference substance solution and need testing solution 5 μ L respectively, injects HPLC-MS instrument, measures, and obtains.
In aforementioned eluent gradient elution process, being changed to of eluent: during beginning, formic acid acetonitrile mutually 2%, aqueous formic acid mutually 98%, stablizes 3min; During 10min, formic acid acetonitrile phase 10%, during aqueous formic acid phase 90%, the 10.1min, formic acid acetonitrile phase 2%, aqueous formic acid phase 98%, stablizes 7min.
Aforementioned Mass Spectrometer Method is multiple-reaction monitoring pattern, reactive ion to and form the required collision energy of stabistor ion and be respectively: gallic acid 169.181 → 125.268, bucking voltage (T μ be Lens Offset) 71V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, protocatechuic acid 152.918 → 109.244, bucking voltage (T μ be Lens Offset) 75V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, interior mark 326.922 → 192.167, bucking voltage (T μ be Lens Offset) 100V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 22eV.
embodiment 2:
Heat-obtaining drenches 8 times of water-soluble solutions for clear particle 1g, be made into aqueous solution, by gastric infusion mode, to rat (265g) heat, drench clear particle aqueous solution 2.65ml, after 60min, rat is taken a blood sample, heparin tube is used liquaemin anti-freezing in advance, by blood after the centrifugal 10min of low speed centrifuge 4000r/min, getting upper plasma is transferred in new blank EP pipe, by acidity, be 2% acidity (acetic acid) acetonitrile-methyl alcohol (3:2) 1:4 protein precipitation 5min by volume, after the even 30s of vortex, 4 ℃ of constant temperature are centrifugal, rotating speed 12000r/min, centrifugal 10min, supernatant liquor is dried up in 40 ℃ in Nitrogen evaporator, residue dissolves with 100 μ l mobile phases, vortex mixes 12000r/min after 60s, 4 ℃ of constant temperature, after centrifugal 10min, get 10 μ l sample introductions, blank plasma is pressed same method and is processed, adopt liquid chromatograph-mass spectrometer to carry out LC-MS/MS by following condition:
Chromatographic condition and testing conditions: chromatographic column is anti-phase C18 chromatographic column, acid (formic acid 0.1%) acetonitrile: acid (formic acid 0.1%) water=0～20:80～100 (volume ratio) are mobile phase, gradient elution, flow velocity is 0.2ml/min, column temperature is 30 ℃, ionization mode is electron spray condition, negative mode monitoring, and spray voltage is 2500V, atomization temperature is 350 ℃, sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, and capillary temperature is 300 ℃;
The preparation of reference substance solution: get gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg, accurately weighed, add pure methanol constant volume to 10ml, obtain mother liquor, working fluid is that mother liquor adds methyl alcohol and is diluted to desired concn.
Determination method: precision is drawn reference substance solution and need testing solution 5 μ L respectively, injects HPLC-MS instrument, measures, and obtains.
In aforementioned eluent gradient elution process, being changed to of eluent: during beginning, formic acid acetonitrile mutually 2%, aqueous formic acid mutually 98%, stablizes 3min; During 10min, formic acid acetonitrile phase 10%, during aqueous formic acid phase 90%, the 10.1min, formic acid acetonitrile phase 2%, aqueous formic acid phase 98%, stablizes 7min.
Aforementioned Mass Spectrometer Method is multiple-reaction monitoring pattern, reactive ion to and form the required collision energy of stabistor ion and be respectively: gallic acid 169.181 → 125.268, bucking voltage (T μ be Lens Offset) 71V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, protocatechuic acid 152.918 → 109.244, bucking voltage (T μ be Lens Offset) 75V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, interior mark 326.922 → 192.167, bucking voltage (T μ be Lens Offset) 100V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 22eV.
embodiment 3:
Heat-obtaining drenches 10 times of water-soluble solutions for clear particle 4g, be made into aqueous solution, by gastric infusion mode, to rat (273g) heat, drench clear particle aqueous solution 2.73ml, after 120min, rat is taken a blood sample, heparin tube is used liquaemin anti-freezing in advance, by blood after the centrifugal 10min of low speed centrifuge 4000r/min, getting upper plasma is transferred in new blank EP pipe, by acidity, be 2% acidity (formic acid/acetic acid=2/1) acetonitrile-methyl alcohol (3:2) 1:4 protein precipitation 5min by volume, after the even 30s of vortex, 4 ℃ of constant temperature are centrifugal, rotating speed 12000r/min, centrifugal 10min, supernatant liquor is dried up in 40 ℃ in Nitrogen evaporator, residue dissolves with 100 μ l mobile phases, vortex mixes 12000r/min after 60s, 4 ℃ of constant temperature, after centrifugal 10min, get 10 μ l sample introductions, blank plasma is pressed same method and is processed, adopt liquid chromatograph-mass spectrometer to carry out LC-MS/MS by following condition:
Chromatographic condition and testing conditions: chromatographic column is anti-phase C18 chromatographic column, acid (formic acid 0.1%) acetonitrile: acid (formic acid 0.1%) water=0～20:80～100 (volume ratio) are mobile phase, gradient elution, flow velocity is 0.2ml/min, column temperature is 30 ℃, ionization mode is electron spray condition, negative mode monitoring, and spray voltage is 2500V, atomization temperature is 350 ℃, sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, and capillary temperature is 300 ℃;
The preparation of reference substance solution: get gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg, accurately weighed, add pure methanol constant volume to 10ml, obtain mother liquor, working fluid is that mother liquor adds methyl alcohol and is diluted to desired concn.
Determination method: precision is drawn reference substance solution and need testing solution 5 μ L respectively, injects HPLC-MS instrument, measures, and obtains.
In aforementioned eluent gradient elution process, being changed to of eluent: during beginning, formic acid acetonitrile mutually 2%, aqueous formic acid mutually 98%, stablizes 3min; During 10min, formic acid acetonitrile phase 10%, during aqueous formic acid phase 90%, the 10.1min, formic acid acetonitrile phase 2%, aqueous formic acid phase 98%, stablizes 7min.
Aforementioned Mass Spectrometer Method is multiple-reaction monitoring pattern, reactive ion to and form the required collision energy of stabistor ion and be respectively: gallic acid 169.181 → 125.268, bucking voltage (T μ be Lens Offset) 71V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, protocatechuic acid 152.918 → 109.244, bucking voltage (T μ be Lens Offset) 75V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV, interior mark 326.922 → 192.167, bucking voltage (T μ be Lens Offset) 100V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 22eV.
Claims (10)
1. heat is drenched two kinds of assay methods that enter blood component of clear particle, and it comprises the following steps:
(1) preparation of test sample:
(a) heat-obtaining drenches clear particle 0.5～4g, adds 5～10 times of water-soluble solutions, gives rat (body weight 230-270g) heat drench clear particle aqueous solution 10ml/kg by gastric infusion mode;
(b), respectively after administration 30～120min, to the rat 0.3～0.5ml that takes a blood sample, by after the centrifugal 5～30min of blood sample 3000～5000r/min rotating speed with liquaemin anti-freezing, get upper plasma and be transferred in new blank EP pipe;
(c) add interior mark Bergenin mother liquor 10 μ l, then by blood plasma and precipitation reagent volume ratio, be 1:4～8 protein precipitation, settling time 2～5min, vortex mixes after 30～60s, under 0～10 ℃ of constant temperature, take the centrifugal 5～15min of 10000～12000r/min (described precipitation reagent be acetonitrile-methyl alcohol (3:2) mixed liquor and add 1%～4% acid, described acid is selected from formic acid, acetic acid or its combination)
(d) upper strata liquid is transferred in new blank EP pipe, in 30～50 ℃ of nitrogen streams, dry up, residue dissolves with 100 μ l mobile phases, and vortex mixes after 30～60s, under 0～10 ℃ of constant temperature, with the centrifugal 5～15min of 10000～12000r/min, get supernatant and be need testing solution;
(e) by step (b) to the method for step (d), the blank plasma of drug administration is not processed, obtained blank solution;
(f) preparation of reference substance solution: precision takes gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg respectively, add separately pure methanol constant volume to 10ml, obtain three kinds of mother liquors, get respectively three kinds of mother liquors appropriate to same measuring bottle, add methyl alcohol and be diluted to desired concn, be reference substance solution;
(2) LC-MS/MS analyzes chromatographic condition and testing conditions:
Chromatographic column is reverse-phase chromatographic column, acid acetonitrile or acidic methanol: acidulous water=0～20:80～100 (volume ratio) is mobile phase, flow velocity is 0.1～0.5mL/min, column temperature is 20～40 ℃, ionization mode is electron spray condition, negative mode monitoring, spray voltage is 2000～3000V, atomization temperature is 300～500 ℃, sheath atmospheric pressure is 30～50mTorr, assisted gas pressure is 5～15mTorr, capillary temperature is 200～400 ℃ (in described acid acetonitrile or acidic methanol or acidulous water, the concentration of acid is 0.05%～0.5%, described acid is selected from formic acid, acetic acid or its combination),
(3) determination method: precision is drawn reference substance solution, blank solution and need testing solution 5～20 μ L respectively, injects HPLC-MS instrument, measures, and obtains.
2. piece the process of claim 1 wherein in step (a), by gastric infusion mode, to rat (230～270g) heat, drench clear particle aqueous solution 2～4ml.
3. piece the process of claim 1 wherein in step (b), respectively at 30min, 60min, after 120min, rat is taken a blood sample.
4. piece the process of claim 1 wherein in step (b), by blood in the centrifugal 10min of low speed centrifuge 4000r/min.
5. piece the process of claim 1 wherein in step (c), is 2% acid acetonitrile-methyl alcohol (3:2) 1:4 protein precipitation 5min by volume by acidity.
6. piece the process of claim 1 wherein that, in step (c), after protein precipitation, after the even 30s of vortex, 4 ℃ of constant temperature are centrifugal, rotating speed 12000r/min, centrifugal 10min.
7. piece the process of claim 1 wherein in step (c), use formic acid: acetic acid=2:1 volume ratio acid mixture carrys out acid precipitation reagent.
8. piece the process of claim 1 wherein in step (d), supernatant liquor is dried up in 40 ℃ in Nitrogen evaporator.
9. piece the process of claim 1 wherein:
In step (d), residue dissolves with 100 μ l mobile phases, and vortex mixes 12000r/min after 60s, and 4 ℃ of constant temperature, get 10 μ l sample introductions after centrifugal 10min;
In step (2), chromatographic column is C18 chromatographic column;
In step (2), in described acid acetonitrile or acidic methanol or acidulous water, the concentration of acid is 0.1%;
In step (2), flow velocity is 0.2ml/min, and column temperature is 30 ℃;
In step (2), ionization mode is electron spray condition, negative mode monitoring, and spray voltage is 2500V, and atomization temperature is 350 ℃, and sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, capillary temperature is 300 ℃;
In step (2), use gradient elution, being changed to of eluent: during beginning, acid acetonitrile mutually 2%, acidic aqueous solution mutually 98%, stablizes 3min; During then to 10min, acid acetonitrile mutually linearity is increased to 10%, acidic aqueous solution phase 90%; During 10.1min, acid acetonitrile changes 2% mutually into, and acidic aqueous solution phase 98%, stablizes 7min;
In step (3), accurate reference substance solution, blank solution and the need testing solution 5 μ L of drawing, inject HPLC-MS instrument respectively; And/or
In step (2), Mass Spectrometer Method is multiple-reaction monitoring pattern, reactive ion to and form the required collision energy of stabistor ion and be respectively: gallic acid 169.181 → 125.268, bucking voltage (T μ be Lens Offset) 71V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV; Protocatechuic acid 152.918 → 109.244, bucking voltage (T μ be Lens Offset) 75V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 13eV; Interior mark Bergenin 326.922 → 192.167, bucking voltage (T μ be Lens Offset) 100V, impact pressure (Collision Press μ re) 1.5mTorr, collision energy (Collision Energy) 22eV.
10. the method for a claim 1, is characterized in that:
Heat-obtaining drenches clear particle 0.5～4g, add 5～10 times of water-soluble solutions, by gastric infusion mode, to rat (230-270g) heat, drench clear particle aqueous solution 10ml/kg, in 30～120min to the rat 0.3～0.5ml that takes a blood sample, be placed in liquaemin anti-freezing test tube, after the centrifugal 5～30min of blood sample 3000～5000r/min rotating speed, getting upper plasma is transferred in new blank EP pipe, add interior mark Bergenin 10 μ l, then press 1%～4% acid acetonitrile-methyl alcohol (3:2) protein precipitation for 1:4～1:8 volume ratio, settling time 2～5min, vortex mixes 10000～12000r/min after 30～60s, 0～10 ℃ of centrifugal 5～15min of constant temperature, upper strata liquid is transferred in new blank EP pipe and dries up in 30～50 ℃ of nitrogen streams, residue dissolves with 100 μ l mobile phases, vortex mixes 10000～12000r/min after 30～60s, 0～10 ℃ of constant temperature, after centrifugal 5～15min, get 5～20 μ l sample introductions, blank plasma is pressed same method and is processed, adopt liquid chromatograph-mass spectrometer to carry out LC-MS/MS analysis by following condition:
Chromatographic condition and testing conditions: chromatographic column is reverse-phase chromatographic column, acid (formic acid or acetic acid 0.05%～0.5%) acetonitrile or methyl alcohol: acid (formic acid or acetic acid 0.05%～0.5%) water=0～20:80～100 (volume ratio) is mobile phase, gradient elution, flow velocity is 0.1～0.5mL/min, column temperature is 20～40 ℃, ionization mode is electron spray condition, negative mode monitoring, spray voltage is 2000～3000V, atomization temperature is 300～500 ℃, sheath atmospheric pressure is 30～50mTorr, and assisted gas pressure is 5～15mTorr, and capillary temperature is 200～400 ℃;
The preparation of reference substance solution: get gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg, accurately weighed, add methanol constant volume to 10ml, obtain mother liquor, working fluid is that mother liquor adds methyl alcohol and is diluted to desired concn;
Or, it is characterized in that: plasma sample operates according to the following steps:
Heat-obtaining drenches clear particle 0.5～4g, add 5～10 times of water-soluble solutions, by gastric infusion mode, to rat (230-270g) heat, drench clear particle aqueous solution 10ml/kg, respectively at 30～120min, afterwards to the rat 0.3～0.5ml that takes a blood sample, by after the centrifugal 10min of blood sample 4000r/min rotating speed with liquaemin anti-freezing, getting upper plasma is transferred in new blank EP pipe, add interior mark Bergenin 10 μ l, then press 2% acid acetonitrile-methyl alcohol (3:2) protein precipitation for 1:4 volume ratio, settling time 5min, vortex mixes the centrifugal 5min of rear 12000r/min, upper strata liquid is transferred in new blank EP pipe and dries up in 40 ℃ of nitrogen streams, residue dissolves with 100 μ l mobile phases, 4 ℃ of constant temperature, after the centrifugal 10min of 12000r/min, get 10 μ l sample introductions, blank plasma is pressed same method and is processed,
Or, it is characterized in that: chromatographic column specification is 2.1 * 150mm, packing material size is 1.7 μ m, take formic acid 0.1% acetonitrile A: formic acid 0.1% water B is mobile phase, press following condition gradient elution: 0～3min2%A, 3.0～3.1min2%A-10%A, 3.1～10.0min10%A, 10.0～10.1min10%-2%A, 10.1～17.0min2%A;
Or, it is characterized in that: mass spectrum spray voltage is 2500V, atomization temperature is 350 ℃, and sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, and capillary temperature is 350 ℃;
Or, it is characterized in that: Mass Spectrometer Method is multiple-reaction monitoring pattern, and reactive ion is to being respectively: gallic acid 169.181 → 125.268, protocatechuic acid 152.918 → 109.244, interior mark 326.922 → 192.167;
Or it preferably includes following steps:
Heat-obtaining drenches 5～10 times of water-soluble solutions for clear particle 0.5～4g, be made into aqueous solution, by gastric infusion mode, to rat (230～270g) heat, drench clear particle aqueous solution 2～4ml, respectively at 30min, 60min, after 120min, rat is taken a blood sample, heparin tube is used liquaemin anti-freezing in advance, by blood after the centrifugal 10min of low speed centrifuge 4000r/min, getting upper plasma is transferred in new blank EP pipe, by acidity, be 2% acid acetonitrile-methyl alcohol (3:2) 1:4 protein precipitation 5min by volume, after the even 30s of vortex, 4 ℃ of constant temperature are centrifugal, rotating speed 12000r/min, centrifugal 10min, supernatant liquor is dried up in 40 ℃ in Nitrogen evaporator, residue dissolves with 100 μ l mobile phases, vortex mixes 12000r/min after 60s, 4 ℃ of constant temperature, after centrifugal 10min, get 10 μ l sample introductions, blank plasma is pressed same method and is processed, adopt liquid chromatograph-mass spectrometer to carry out LC-MS/MS by following condition:
Chromatographic condition and testing conditions: chromatographic column is reverse-phase chromatographic column, acid (formic acid 0.1%) acetonitrile: acid (formic acid 0.1%) water=0～20:80～100 (volume ratio) are mobile phase, gradient elution, flow velocity is 0.2ml/min, column temperature is 30 ℃, ionization mode is electron spray condition, negative mode monitoring, and spray voltage is 2500V, atomization temperature is 350 ℃, sheath atmospheric pressure is 40mTorr, and assisted gas pressure is 10mTorr, and capillary temperature is 300 ℃;
The preparation of reference substance solution: get gallic acid reference substance, protocatechuic acid reference substance, Bergenin reference substance 0.01～1mg, accurately weighed, add pure methanol constant volume to 10ml, obtain mother liquor, working fluid is that mother liquor adds methyl alcohol and is diluted to desired concn;
Determination method: precision is drawn reference substance solution and need testing solution 5 μ L respectively, injects HPLC-MS instrument, measures, and obtains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410234840.4A CN103983732B (en) | 2014-05-29 | 2014-05-29 | Determination method for two components in plasma of Relinqing granules |
Applications Claiming Priority (1)
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| CN108982735A (en) * | 2018-06-29 | 2018-12-11 | 无锡济民可信山禾药业股份有限公司 | Borneol and Determination of Muscone method in a kind of XINGNAOJING ZHUSHEYE blood |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108732259A (en) * | 2018-02-08 | 2018-11-02 | 贵州医科大学 | Enter the detection method of blood chemistry ingredient in Polygonum orientale flower extract |
| CN108982735A (en) * | 2018-06-29 | 2018-12-11 | 无锡济民可信山禾药业股份有限公司 | Borneol and Determination of Muscone method in a kind of XINGNAOJING ZHUSHEYE blood |
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Denomination of invention: Determination of two kinds of blood components in Relinqing granules Effective date of registration: 20230223 Granted publication date: 20150513 Pledgee: Agricultural Bank of China Limited by Share Ltd. Guiyang Xinhua Branch Pledgor: GUIZHOU WARMEN PHARMACEUTICAL Co.,Ltd. Registration number: Y2023990000128 |
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