CN108181400A - The method that UHPLC-QQQ-MS/MS measures 6 kinds of chemical composition contents in spina date seed decocting liquid simultaneously - Google Patents
The method that UHPLC-QQQ-MS/MS measures 6 kinds of chemical composition contents in spina date seed decocting liquid simultaneously Download PDFInfo
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- CN108181400A CN108181400A CN201810071306.4A CN201810071306A CN108181400A CN 108181400 A CN108181400 A CN 108181400A CN 201810071306 A CN201810071306 A CN 201810071306A CN 108181400 A CN108181400 A CN 108181400A
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- date seed
- spina date
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- decocting liquid
- spinosin
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- 239000000126 substance Substances 0.000 title claims abstract description 36
- 239000000203 mixture Substances 0.000 title claims abstract description 34
- 239000007788 liquid Substances 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000004885 tandem mass spectrometry Methods 0.000 title claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- VGGSULWDCMWZPO-ODEMIOGVSA-N spinosin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=2C(=O)C=C(OC=2C=C1OC)C=1C=CC(O)=CC=1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VGGSULWDCMWZPO-ODEMIOGVSA-N 0.000 claims description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- VGGSULWDCMWZPO-UHFFFAOYSA-N flavoayamenin Natural products COC1=CC=2OC(C=3C=CC(O)=CC=3)=CC(=O)C=2C(O)=C1C1OC(CO)C(O)C(O)C1OC1OC(CO)C(O)C(O)C1O VGGSULWDCMWZPO-UHFFFAOYSA-N 0.000 claims description 27
- 239000013558 reference substance Substances 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 19
- 239000012085 test solution Substances 0.000 claims description 19
- 239000007789 gas Substances 0.000 claims description 15
- -1 acyl spinosin Chemical compound 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- SMRPGWBDLOQHOS-UHFFFAOYSA-N 5-[4,5-dihydroxy-6-(hydroxymethyl)-3-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxy-3,4-dihydroxy-6-[[9-hydroxy-4-(hydroxymethyl)-4,6a,6b,8a,11,11,14b-heptamethyl-14-oxo-2,3,4a,5,6,7,8,9,10,12,12a,14a-dodecahydro-1H-picen-3-yl]oxy]oxane-2-carboxylic acid Chemical compound OC1C(O)C(O)C(C)OC1OC1C(OC2C(OC(C(O)C2O)C(O)=O)OC2C(C3C(C4C(C5(CCC6(C)C(O)CC(C)(C)CC6C5=CC4=O)C)(C)CC3)(C)CC2)(C)CO)OC(CO)C(O)C1O SMRPGWBDLOQHOS-UHFFFAOYSA-N 0.000 claims description 13
- PPRSVUXPYPBULA-UHFFFAOYSA-N saponin A Natural products CC1(C)CCC2(CCC3(C)C(=CCC4C5(C)CCC(OC6OC(CO)C(O)C(O)C6=O)C(C)(C)C5CCC34C)C2C1)C(=O)O PPRSVUXPYPBULA-UHFFFAOYSA-N 0.000 claims description 13
- QGJZLNKBHJESQX-UHFFFAOYSA-N 3-Epi-Betulin-Saeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C(=C)C)C5C4CCC3C21C QGJZLNKBHJESQX-UHFFFAOYSA-N 0.000 claims description 11
- QGJZLNKBHJESQX-FZFNOLFKSA-N betulinic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C QGJZLNKBHJESQX-FZFNOLFKSA-N 0.000 claims description 11
- 238000009835 boiling Methods 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- JMJXWSOEKNACTH-UHFFFAOYSA-N camelliin B Natural products OC1OC(C2OC(=O)C=3C(=C(O)C(O)=C(O)C=3)C3=C(O)C=4O)COC(=O)C3=CC=4OC3=C(O)C(O)=C(O)C=C3C(=O)OC(C(C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C3OC(=O)C4=C5)OC(=O)C=6C=C(O)C(O)=C(O)C=6)OC3COC(=O)C3=CC(O)=C(O)C(O)=C3C4=C(O)C(O)=C5OC3=C(O)C(O)=C(O)C=C3C(=O)OC1C2OC(=O)C1=CC(O)=C(O)C(O)=C1 JMJXWSOEKNACTH-UHFFFAOYSA-N 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000004949 mass spectrometry Methods 0.000 claims description 6
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 238000000889 atomisation Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 abstract description 11
- 238000003556 assay Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 238000003908 quality control method Methods 0.000 abstract description 2
- 238000001195 ultra high performance liquid chromatography Methods 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 12
- 239000012071 phase Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 244000274847 Betula papyrifera Species 0.000 description 3
- 235000009113 Betula papyrifera Nutrition 0.000 description 3
- 235000009109 Betula pendula Nutrition 0.000 description 3
- 235000010928 Betula populifolia Nutrition 0.000 description 3
- 235000002992 Betula pubescens Nutrition 0.000 description 3
- 240000008866 Ziziphus nummularia Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 229930182490 saponin Natural products 0.000 description 3
- 150000007949 saponins Chemical class 0.000 description 3
- 235000017709 saponins Nutrition 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 240000001548 Camellia japonica Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 235000018597 common camellia Nutrition 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 150000002825 nitriles Chemical class 0.000 description 2
- 150000003648 triterpenes Chemical class 0.000 description 2
- RSWGJHLUYNHPMX-UHFFFAOYSA-N 1,4a-dimethyl-7-propan-2-yl-2,3,4,4b,5,6,10,10a-octahydrophenanthrene-1-carboxylic acid Chemical compound C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-UHFFFAOYSA-N 0.000 description 1
- RANQPHKSRUUPKK-UHFFFAOYSA-N 3-O-[alpha-L-arabinofuranosyl-(1->2)-O-{beta-D-glucopyranosyl-(1->3)}-(alpha-L-arabinopyranosyl)]jujubogenin Natural products CC(C)=CC1CC(C)(O)C2C3CCC4C5(C)CCC(OC6OCC(O)C(OC7OC(CO)C(O)C(O)C7O)C6OC6OC(CO)C(O)C6O)C(C)(C)C5CCC4(C)C33COC2(C3)O1 RANQPHKSRUUPKK-UHFFFAOYSA-N 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 241000219100 Rhamnaceae Species 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229930192286 jujuboside Natural products 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 229940125725 tranquilizer Drugs 0.000 description 1
- 239000003204 tranquilizing agent Substances 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Steroid Compounds (AREA)
Abstract
The present invention relates to a kind of methods for measuring 6 kinds of chemical composition contents in spina date seed decocting liquid simultaneously using UHPLC QQQ MS/MS technologies, belong to traditional Chinese medicine ingredients analysis field.The present invention establish and meanwhile measure 6 kinds of chemical composition contents in spina date seed decocting liquid analysis method specificity it is strong, quick, sensitive and accurately and reliably, compound quality number information can be accurately provided while assay is completed to achieve the effect that Components identification, this method can provide technical support for spina date seed clinical practice and quality control.
Description
Technical field
The present invention relates to it is a kind of using UHPLC-QQQ-MS/MS technologies measure simultaneously in spina date seed decocting liquid 6 kinds of chemistry into
Divide the method for content, belong to traditional Chinese medicine ingredients analysis field.
Background technology
Spina date seed is as heart-nourishing tranquilizer common on tcm clinical practice, from the drying and ripening kind of rhamnaceae plant wild jujube
Son.Though there is scholar to be studied the assay of chemical composition in spina date seed at present, in view of containing Huang in spina date seed
The numerous compositions such as ketone, saponins, alkaloids and triterpenes, and by the detector performance of traditional sensing techniques HPLC-DAD
Limitation quickly, accurately, delicately to the chemical composition of plurality of classes in spina date seed can not carry out assay simultaneously.In addition, by
It focuses mostly in existing research in using organic solvent, as Extraction solvent, there are larger differences with spina date seed clinical practice pattern for this
Not, result of study can be that the reference value that clinical practice provides is limited.
Invention content
Above-mentioned in order to solve the problems, such as, the present invention is established a kind of for wild jujube using UHPLC-QQQ-MS/MS technologies
The method of 6 kinds of different classes of chemical compositions assay simultaneously, this 6 kinds of chemical compositions cover Huang in structure in benevolence decocting liquid
Ketone, saponins and triterpenes, this enable established content assaying method comprehensively, it is accurate, in depth control spina date seed
Quality.
Invention adopts the following technical scheme that：
1. the preparation of test solution：
According to spina date seed Clinical Experience, precision weighs spina date seed 10g, adds 10 times of water in decocting medicine pot, impregnates
30min after boiling by intense fire, keeps slight boiling condition to decoct 1h, liquid is filtered out with 4 layers of gauze；The dregs of a decoction add 8 times of water in decocting medicine pot again
In, after boiling with high heat, slight boiling condition is kept to decoct 1h, filter out liquid with 4 layers of gauze, merge decoction liquor twice, add appropriate amount of water
200ml is settled to, obtains spina date seed decocting liquid sample.
Precision pipettes spina date seed decocting liquid sample 0.2ml and puts in 5ml measuring bottles, addition methanol constant volume to 5ml, ultrasonic 10min,
It lets cool, takes upper solution appropriate, 12000r/min centrifugation 10min, Aspirate supernatant is as test solution；
2. the preparation of mixed reference substance solution：
Precision weighs spinosin, 6 " respectively '-asafoetide acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellia
Glycosides B, betulic acid reference substance are appropriate, add in methanol and dissolve and dilute, shake up, it is respectively 6.312 μ g/ml, 5.885 to obtain concentration
μ g/ml, 0.991 μ g/ml, 0.668 μ g/ml, 0.640 μ g/ml, 4.504 μ g/ml mixed reference substance solution；
3. measuring method：
The concentration of 6 kinds of chemical compositions in mixing reference substance and test solution is measured using UHPLC-QQQ-MS/MS methods.
Chromatographic condition：Chromatographic column is Agilent C18(2.1mm × 100mm, 1.8 μm) reverse-phase chromatographic column, A (water)-B (second
Nitrile) as mobile phase progress gradient elution, elution program is：0-1.2min, the volume fraction of acetonitrile are linearly increasing to from 5%
31%, 1.2-2.5min, the volume fraction of acetonitrile are linearly increasing to 95%, 2.5-4.5min, the volume fraction of acetonitrile from 31%
100%, 4.5-5min is linearly increasing to from 95%, the volume fraction of acetonitrile is maintained at 100%.Flow velocity：0.3ml/min；Column temperature
40 DEG C, sample size：1μl.
Mass Spectrometry Conditions：Using ESI-Pattern is detected, and scan mode uses multiple-reaction monitoring pattern (MRM).Parameter is such as
Under：550 DEG C of ion source temperature；Atomization gas 55psi；Auxiliary heating gas 55psi；Gas curtain gas 35psi；Nitrogen is as spraying auxiliary gas
Body.Each ingredient (spinosin, 6 " '-asafoetide acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellin B, white birch fat
Acid) mass spectrometry parameters Q1/Q3 (m/z) be respectively：607.5/427.1、783.2/427.4、1205.9/1073.9、1043.6/
911.5、725.2/284.0、455.3/455.3；Go cluster voltage (DP) be respectively -26.46V, -25.83V, -60.00V, -
27.20V、-23.72V、-97.00V；Collision energy (CE) is respectively：-41.60V、-50.92V、-60.00V、-49.77V、-
53.07V、-20.00V。
3 measuring method of above-mentioned steps includes the measure of linear relationship：Precision draws mixed reference substance solution, dilute according to multiple
Interpretation of the law is diluted, and the control series product for obtaining 6 different quality concentration that extension rate is respectively 1,2,4,8,16,32 times are molten
Liquid measures respectively under test conditions, the chromatographic peak area of 6 kinds of chemical compositions is recorded, using reference substance mass concentration as abscissa
(X), peak area is ordinate (Y), draws standard curve, and carry out linear regression, obtains calibration curve equation.
3 measuring method of above-mentioned steps includes the measure of precision：Precision draws mixed reference substance solution, under test conditions
Continuous sample introduction 6 times records the peak area of 6 kinds of chemical compositions.
3 measuring method of above-mentioned steps includes the measure of repeatability：It takes with a collection of Spine Date Seed sample, it is molten according to test sample
The preparation method of liquid is parallel to prepare 6 parts of test solutions, is measured respectively under test condition, records the peak face of 6 kinds of chemical compositions
Product.
3 measuring method of above-mentioned steps includes the measure of stability：Test solution is taken, is existed in 0,2,4,8,12,24,48h
It is measured respectively under test condition, records the peak area of 6 kinds of chemical compositions.
3 measuring method of above-mentioned steps includes sample recovery rate experiment：Precision is drawn under " preparation of test solution " item
Know the spina date seed decocting liquid sample 0.1ml of content, put in 5ml measuring bottles, it is accurate respectively to add in certain density reference substance solution (this
Pi Nuosu, 6 " '-asafoetide acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellin B, betulic acid concentration are respectively
157.564th, 73.535,12.522,8.042,8.076,28.029 μ g/ml) 0.1ml, it adds in proper amount of methanol and is settled to 5ml, surpass
Sound 10min, lets cool, and supernatant 12000r/min is taken to centrifuge 10min, and Aspirate supernatant parallel 6 parts, is being surveyed as test solution
It is measured under the conditions of examination, records the peak area of 6 kinds of chemical compositions, calculate the sample recovery rate of this method.
It is an advantage of the invention that：1. the experimental results showed that under the test condition of optimization, surveyed in spina date seed decocting liquid
6 kinds of chemical compositions can efficiently separate under the condition of gradient elution of water-acetonitrile system in can reach in short 5min, survey
Period for determining method is short, separative efficiency is high；2. mass spectrum has highly sensitive and strong selectivity, can be in Accurate Determining component content
While, the mass number information of ingredient is accurately provided, achievees the effect that while compound is differentiated；3. the present invention is to wild jujube
6 kinds of different classes of chemical compositions carry out Simultaneous Quantitative Analysis, high-specificity, easy to operate, repeated good, knot in benevolence decocting liquid
Fruit accurately and reliably, can provide technical support and theoretical reference for spina date seed clinical practice and quality control.
Description of the drawings
Fig. 1 is the extraction ion stream mass chromatogram of 6 kinds of chemical compositions in mixed reference substance solution of the present invention, wherein
A：Spinosin；B：6 " '-asafoetide acyl spinosin；C：Saponin A；D：Spine Date Seed jujubosideB；E：Camellin
B；F：Betulic acid.
Fig. 2 is the chemical structural formula figure of 6 kinds of ingredients to be measured in test solution of the present invention, wherein
A：Spinosin；B：6 " '-asafoetide acyl spinosin；C：Saponin A；D：Spine Date Seed jujubosideB；E：Camellin
B；F：Betulic acid.
Specific implementation method
Form by the following examples is described in further detail, but should not be by this above of present invention again
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to following example, and all technologies realized based on the above of the present invention are equal
Belong to the scope of the present invention.
Embodiment 1：The method that UHPLC-QQQ-MS/MS measures 6 kinds of chemical composition contents in spina date seed decocting liquid simultaneously：
1. medicine materical crude slice, instrument and reagent
1.1 medicine materical crude slice sources
Spina date seed is purchased from Tongling, Anhui Province He Tian medicine materical crude slice Co., Ltd (Tongling, China)；Through pharmaceutical college of Nanjing University of Traditional Chinese Medicine
Professor Chen Jianwei identifies, is certified products.
1.2 instrument
(Shimadzu LC 30AD are pumped Shimadzu highly effective liquid phase chromatographic systems, SIL 30AC autosamplers, 30AC columns
Incubator)；5500 mass spectrographs of Triple Quadrupole (American AB Sciex companies) ESI ion sources；Mass spectrum work station：
Analyst TF1.6；Ultrasonic cleaner (KQ-500, Kunshan Ultrasonic Instruments Co., Ltd.)；285 electronic balances of AG
(METTLER TOLEDO, d=0.01mg)；Pure water meter (Nanjing Yi Puyida Development Co., Ltds, model：EPED-10TF is compiled
Number：0903090818)；Supercentrifuge (Changsha Xiang Zhi centrifuges Instrument Ltd., model：XZ-6G).
1.3 reagent
Spinosin (lot number S-043-150626) (purity>98%), 6 " '-asafoetide acyl spinosin (lot number A-
011-170731) (purity>98%), Saponin A (lot number S-045-160809) (purity>98%), Spine Date Seed jujubosideB
(lot number S-046-151205) (purity>98%) reference substance is purchased from Chengdu Rui Fensi Co., Ltds；(lot number is camellin B
170313-080) (purity>98%), betulic acid (lot number is JYB 201701) (purity>98%) reference substance is purchased from Nanjing gold
Beneficial one hundred bio tech ltd.
Acetonitrile is pure (Merck companies of the U.S.) for mass spectrum, makes ultra-pure water by oneself.
2. method and result
The preparation of 2.1 mixed reference substance solutions
Precision weighs spinosin, 6 " respectively '-asafoetide acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellia
Glycosides B, betulic acid reference substance are appropriate, add in methanol and dissolve and dilute, shake up, it is respectively 6.312 μ g/ml, 5.885 to obtain concentration
μ g/ml, 0.991 μ g/ml, 0.668 μ g/ml, 0.640 μ g/ml, 4.504 μ g/ml mixed reference substance solution；
The preparation of 2.2 test solutions
According to spina date seed Clinical Experience, precision weighs spina date seed 10g, adds 10 times of water in decocting medicine pot, impregnates
30min after boiling by intense fire, keeps slight boiling condition to decoct 1h, liquid is filtered out with 4 layers of gauze；The dregs of a decoction add 8 times of water in decocting medicine pot again
In, after boiling with high heat, slight boiling condition is kept to decoct 1h, filter out liquid with 4 layers of gauze, merge decoction liquor twice, add appropriate amount of water
200ml is settled to, is spina date seed decocting liquid sample.Precision pipettes spina date seed decocting liquid sample 0.2ml and puts in 5ml measuring bottles, adds in
Methanol constant volume is let cool to 5ml, ultrasonic 10min, takes upper solution appropriate, and 12000r/min centrifugation 10min, Aspirate supernatant is made
For test solution；
2.3 chromatographic conditions and mass spectrometry parameters condition
Chromatographic condition：Chromatographic column is Agilent C18(2.1mm × 100mm, 1.8 μm) reverse-phase chromatographic column, A (water)-B (second
Nitrile) as mobile phase progress gradient elution, elution program is：0-1.2min, the volume fraction of acetonitrile are linearly increasing to from 5%
31%, 1.2-2.5min, the volume fraction of acetonitrile are linearly increasing to 95%, 2.5-4.5min, the volume fraction of acetonitrile from 31%
100%, 4.5-5min is linearly increasing to from 95%, the volume fraction of acetonitrile is maintained at 100%.Flow velocity：0.3ml/min；Column temperature
40 DEG C, sample size：1μl.
Mass Spectrometry Conditions：Using ESI-Pattern is detected, and scan mode uses multiple-reaction monitoring pattern (MRM).Parameter is such as
Under：550 DEG C of ion source temperature；Atomization gas 55psi；Auxiliary heating gas 55psi；Gas curtain gas 35psi；Nitrogen is as spraying auxiliary gas
Body.Each ingredient (spinosin, 6 " '-asafoetide acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellin B, white birch fat
Acid) mass spectrometry parameters Q1/Q3 (m/z) be respectively：607.5/427.1、783.2/427.4、1205.9/1073.9、1043.6/
911.5、725.2/284.0、455.3/455.3；Go cluster voltage (DP) be respectively -26.46V, -25.83V, -60.00V, -
27.20V、-23.72V、-97.00V；Collision energy (CE) is respectively：-41.60V、-50.92V、-60.00V、-49.77V、-
53.07V、-20.00V。
2.4 linear relationships are investigated
Precision draws mixed reference substance solution, is diluted according to multiple dilution method, obtain extension rate be respectively 1,2,4,
8th, the control series product solution of 16,32 times of 6 different quality concentration, measures respectively under test conditions, record 6 kinds of chemistry into
The chromatographic peak area divided, using reference substance mass concentration as abscissa (X), peak area is ordinate (Y), draws standard curve, and
Linear regression is carried out, calibration curve equation is obtained, the results are shown in Table 1.The result shows that 6 kinds of chemical compositions are linear in the range of setting
Relationship is good.
Linear equation, related coefficient and the range of linearity of 6 kinds of chemical compositions in 1 spina date seed of table
2.5 precision test
Precision draws reference substance solution, the continuous sample introduction 6 times under the conditions of " 2.3 ", the peak face of 6 kinds of chemical compositions of record
Product.The results show that spinosin, 6 " '-asafoetide acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellin B, white birch
The RSD (n=6) of resin acid peak area is respectively 0.60%, 2.51%, 2.75%, 2.91%, 1.12%, 2.62%, shows instrument
Precision is good.
2.6 repetitive test
It takes with a collection of spina date seed medicine materical crude slice, the parallel obtained test sample of method prepared according to test solution under " 2.2 " is molten
6 parts of liquid is measured under the conditions of " 2.3 ", calculates the concentration of each chemical composition.The results show that spinosin, 6 " '-asafoetide
Acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellin B, betulic acid mean concentration be respectively 3.807,
1.451、0.225、0.186、0.101、0.863μg/ml；RSD is respectively 1.20%, 2.64%, 2.60%, 2.21%,
2.98%th, 1.18%, show that the repeatability of method is good.
2.7 stability test
Test solution is taken, is placed at room temperature for, condition measures respectively under 0,2,4,8,12,24,48h are according to " 2.3 ", note
Record the peak area of 6 kinds of chemical compositions.The results show that spinosin, 6 " '-asafoetide acyl spinosin, Saponin A, spina date seed
Saponin(e B, camellin B, betulic acid peak area RSD be respectively 2.96%, 2.53%, 2.82%, 2.10%, 2.31%,
2.72%, show that test sample is being placed at room temperature for stabilization in 48h.
2.8 sample recovery rates are tested
Precision draws the spina date seed decocting liquid sample 0.1ml of known content under " preparation of test solution " item, puts 5ml amounts
It is accurate respectively to add in certain density reference substance solution (spinosin, 6 " '-asafoetide acyl spinosin, jujuboside in bottle
A, Spine Date Seed jujubosideB, camellin B, betulic acid) 0.1ml, it adds in proper amount of methanol and is settled to 5ml, ultrasonic 10min lets cool, takes
Supernatant 12000r/min centrifuges 10min, and Aspirate supernatant is surveyed under test conditions as test solution, parallel 6 parts of preparation
It is fixed, the peak area of 6 kinds of chemical compositions is recorded, calculates the sample recovery rate of this method.It the results are shown in Table 2.6 kinds of chemical constituents determinations
The rate of recovery is in the range of 95.00~105.00%, RSD≤3.0%, shows that method is accurate, reliable.
The rate of recovery (n=6) of 26 kinds of chemical compositions of table
2.9 sample sizes measure
Spina date seed pharmaceutical decocting piece 10 batches is taken, prepares test solution according to test solution preparation method under " 2.2 ", often
3 parts of criticize flat row, under the conditions of " 2.3 ", sample introduction is analyzed, records the peak area of 6 kinds of chemical compositions and calculates chemical composition and contain
Amount, the results are shown in Table 3.
The assay result (μ g/g) of 6 kinds of chemical compositions in 3 10 batches of spina date seed decocting liquids of table
3. it discusses
The content of 6 kinds of chemical compositions in spina date seed decocting liquid is analyzed using UHPLC-QQQ-MS/MS methods, is considered not
The structure of generic ingredient and polarity feature, in ESI-It is detected under ion mode, using MRM mode monitorings, each ingredient exists
Different ion channels has preferable response；Mobile phase is optimized to realize that each ingredient efficiently separates, compared
Machine phase (methanol/acetonitrile) and water phase (water/0.1% formic acid water), finally found that using acetonitrile-water as flow phase system progress
During gradient elution, 6 kinds of chemical compositions, which can be realized, efficiently separates and obtains good peak shape, and the retention time of each ingredient is significantly
Shorten, which improves conventional efficient, saves reagent, has practical economic implications.It is investigated in solvent selection
Methanol, 60% methanol solution and 60% ethanol solution, finally found that methanol effectively can dissolve in spina date seed decocting liquid 6 kinds into
Point, therefore solvent uses methanol.
4. conclusion
The method that the present invention is established is easy to operate, and 6 kinds of chemical compositions obtain good under conditions of being selected herein
Separating effect and response.The measure analysis of component content can be completed in the present invention in 5min, and measurement result is accurate, stability
Well, it is a kind of reliable method for measuring various structures type chemical ingredient in spina date seed decocting liquid, can is the comprehensive matter of spina date seed
Amount control provides experimental basis with evaluation.
Claims (1)
1. a kind of method for measuring 6 kinds of chemical composition contents in spina date seed decocting liquid simultaneously using UHPLC-QQQ-MS/MS, special
Sign is to include the following steps：
(1) preparation of mixed reference substance solution：
Respectively precision weigh spinosin, 6 " '-asafoetide acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellin B,
Betulic acid reference substance is appropriate, adds in methanol and dissolves and dilute, shakes up, it is respectively 6.312 μ g/ml, 5.885 μ g/ to obtain concentration
Ml, 0.991 μ g/ml, 0.668 μ g/ml, 0.640 μ g/ml, 4.504 μ g/ml mixed reference substance solution；
(2) preparation of test solution：
According to spina date seed Clinical Experience, precision weighs spina date seed 10g, adds 10 times of water in decocting medicine pot, impregnates 30min, military
After fire boils, slight boiling condition is kept to decoct 1h, liquid is filtered out with 4 layers of gauze；The dregs of a decoction add 8 times of water in decocting medicine pot again, with high heat
After boiling, slight boiling condition is kept to decoct 1h, filter out liquid with 4 layers of gauze, merge decoction liquor twice, appropriate amount of water is added to be settled to
200ml obtains spina date seed decocting liquid sample；
Precision pipettes spina date seed decocting liquid sample 0.2ml and puts in 5ml measuring bottles, and addition methanol constant volume to 5ml, ultrasonic 10min is put
It is cold, take upper solution appropriate, 12000r/min centrifugation 10min, Aspirate supernatant is as test solution；
(3) measuring method：
The concentration of 6 kinds of chemical compositions in mixing reference substance and test solution is measured using UHPLC-QQQ-MS/MS methods；
Chromatographic condition：Chromatographic column is Agilent C18, 2.1mm × 100mm, 1.8 μm, reverse-phase chromatographic column, A water-B acetonitriles are as stream
Dynamic mutually to carry out gradient elution, elution program is：0-1.2min, the volume fraction of acetonitrile are linearly increasing to 31%, 1.2- from 5%
2.5min, the volume fraction of acetonitrile are linearly increasing to 95%, 2.5-4.5min from 31%, and the volume fraction of acetonitrile is linear from 95%
100%, 4.5-5min is increased to, the volume fraction of acetonitrile is maintained at 100%.Flow velocity：0.3ml/min；40 DEG C of column temperature, sample introduction
Amount：1μl；
Mass Spectrometry Conditions：Using ESI-Pattern is detected, and scan mode uses multiple-reaction monitoring pattern MRM；Parameter is as follows：Ion
550 DEG C of source temperature；Atomization gas 55psi；Auxiliary heating gas 55psi；Gas curtain gas 35psi；Nitrogen is as spraying auxiliary gas；Respectively into
The matter of spinosin, 6 " '-asafoetide acyl spinosin, Saponin A, Spine Date Seed jujubosideB, camellin B, betulic acid in point
Composing parameter Q1/Q3 is respectively：607.5/427.1、783.2/427.4、1205.9/1073.9、1043.6/911.5、725.2/
284.0、455.3/455.3；Go cluster voltage DP be respectively -26.46V, -25.83V, -60.00V, -27.20V, -23.72V, -
97.00V；Collision energy CE is respectively：-41.60V、-50.92V、-60.00V、-49.77V、-53.07V、-20.00V.
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| CN109324126A (en) * | 2018-09-21 | 2019-02-12 | 山西中医药大学 | A method of 9 kinds of chemical components in semen ziziphi spinosae are measured simultaneously using UPLC-MS/MS |
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| CN109632995A (en) * | 2018-12-21 | 2019-04-16 | 广东方制药有限公司 | A kind of method for building up of semen ziziphi spinosae flavones ingredient UPLC finger-print and application |
| CN109632995B (en) * | 2018-12-21 | 2022-02-11 | 广东一方制药有限公司 | Establishing method and application of UPLC fingerprint spectrum of spina date seed flavonoid component |
| CN114487181A (en) * | 2022-01-20 | 2022-05-13 | 广西壮族自治区食品药品检验所 | Method for measuring content of jujuboside A and saponin B in Tianwang heart tonifying preparation |
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