CN103149301A - Method for simultaneously determining contents of 10 nucleoside components in sowthistle-leaf ixeris seedling injection by utilizing HPLC-DAD (High Performance Liquid Chromatography-Diode Array detector) method - Google Patents
Method for simultaneously determining contents of 10 nucleoside components in sowthistle-leaf ixeris seedling injection by utilizing HPLC-DAD (High Performance Liquid Chromatography-Diode Array detector) method Download PDFInfo
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- 241001412304 Ixeris Species 0.000 title abstract description 8
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- 150000003833 nucleoside derivatives Chemical class 0.000 title abstract 3
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- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims description 64
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- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims description 55
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Abstract
The invention relates to a method for simultaneously determining contents of 10 nucleoside components in sowthistle-leaf ixeris seedling injection by utilizing an HPLC-DAD (High Performance Liquid Chromatography-Diode Array detector) method, and belongs to the field of traditional Chinese medicine component analysis. The method comprises the following steps of: (1) preparation of a mixed reference liquid; (2) preparation of a test liquid; and (3) determining method: precisely absorbing the mixed reference liquid and the test liquid respectively by the HPLC-DAD method, injecting to a detector and detecting, wherein the chromatographic condition is as follows: the chromatographic column is DiamonsilC18(2), the detection wavelength is 260nm, the flow rate is 1mL/min, the column temperature is 35 DEG C, the sample size is 10 microlitres, and the gradient elute condition is 0-5 minutes; performing gradient eluting based on 2% of acetonitrile by volume fraction; linearly increasing to 9% of acetonitrile with 5-9 minutes; linearly increasing to 11% of acetonitrile with 9-15 minutes; and linearly increasing to 40% of acetonitrile with 15-25 minutes. The method for simultaneously determining the contents of the 10 nucleoside components in sowthistle-leaf ixeris seedling injection established by the invention is simple in analytic method, good in repeatability and accurate and reliable, and can provide reference for quality control of the sowthistle-leaf ixeris seedling injection.
Description
Technical field
The present invention relates to a kind of HPLC-DAD method and measure simultaneously the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection.Belong to the traditional Chinese medicine ingredients analysis field.
Background technology
In the prior art, sowthistle-leaf ixeris seedling another name babysbreath, denticulate ixeris herb, mainly be distributed in the ground such as China northeast and the Inner Mongol, living dry herb then for feverfew irexis sonchifolia (Ixeris sonchifolia (Bge.) Hance), cold in nature, bitter, suffering have effect clearing heat and detoxicating, the apocenosis pain relieving
[1]Sowthistle-leaf ixeris seedling (dish arteries and veins spirit) parenteral solution is to extract the refining intravenous fluid that forms take sowthistle-leaf ixeris seedling as raw material, has promoting blood circulation and removing blood stasisly, improves microcirculatory effect
[2]This parenteral solution has gone on the market for many years, determined curative effect.Experimental study shows, Ixeris Sonchifolia Hance injection chemical composition more complicated, and the main active relevant to its clinical efficacy is ucleosides, organic acid and flavones ingredient.
Ixeris Sonchifolia Hance injection (former name: Diemailing injection) list national drug standards product (the accurate word Z20025450 of traditional Chinese medicines) in, produced by Tonghua Huaxia Pharmaceutical Co., Ltd..
Prescription: irexis sonchifolia 1000g, make 1000ml.
method for making: get irexis sonchifolia, the boiling secondary, 1 hour for the first time, 0.5 hour for the second time, collecting decoction, be concentrated into every 1ml and be equivalent to crude drug in whole 0.5g, let cool to below 40 ℃, under agitation add 10% calcium oxide breast and regulate pH value to 10, placed 12 hours, centrifugal, centrifugal sediment is weighed, be suspended in 5.3 times of amount 95% ethanol (make contain alcohol amount reach 80%), add 50% sulfuric acid solution and regulate the pH value to 3-4, fully stir to make and react completely, centrifugal, centrifugate adds in 40% sodium hydroxide solution and pH value to 7.0, filter, filtrate recycling ethanol, and wave most ethanol, be diluted to every 1ml with water for injection and be equivalent to crude drug in whole 4g, putting below-5 ℃ places more than 12 hours, filter, filtrate adds 0.1-0.2% medical active carbon powder, boiled 15 minutes, putting below-5 ℃ places more than 24 hours, filter, filtrate injecting is diluted with water to ormal weight, regulate the pH value to 7.0-7.5, filter, embedding, sterilization, and get final product.Proterties: this product is that light yellowish brown is to the clear liquid of yellowish-brown.
Assay: the every 10ml of this product contains general flavone with anhydrous rutin (C
27H
30O
16) meter, must not be less than 4.0mg.Contain irexis sonchifolia with adenosine (C
10H
12N
5O
4) meter, must not be less than 0.025mg.
Function cures mainly: promoting blood circulation and stopping pain, Qingre Quyu.Be used for the obstruction of qi in the chest of hemostasis impatency, card is seen: uncomfortable in chest, pained, and bitter taste, tongue is dark red or deposit ecchymosis etc.Be applicable to coronary heart diseases and angina pectoris and see above-mentioned symptom person.The person that also can be used for the cerebral infarction.
Usage and dosage: drip-feed, a 10-40ml, 1 time on the one; Use after being diluted to 250-500ml with 5% glucose or 0.9% sodium chloride injection; 14 days is a course for the treatment of; Or follow the doctor's advice.
Specification: every dress (1) 10ml, (2) 20ml, (3) 40ml.
Experimental study shows, Ixeris Sonchifolia Hance injection chemical composition more complicated, and the main active relevant to its clinical efficacy is ucleosides, organic acid and flavones ingredient.
Gradient elution is the sustain life basic composition element of activity of biological cell, participates in the DNA metabolic process, by the multiple physiological action of the performance of the purinoceptor in human activin.(cytidine) has the effects such as antitumor and antiviral as cytidine; It is synthetic that uridine (uridine) participates in glycogen, helps to improve the hypoxia-bearing capability of cell, improves the body antibody horizontal, and it is synthetic with viral gene to block cancer cell; Guanosine (guanosine) has immunoregulatory physiologically active; Particularly adenosine (adenosine) has and resists myocardial ischemia, anticoagulation, anti-inflammatory, improves the heart and brain blood circulation, prevents arrhythmia cordis, suppresses neurotransmitter and discharge and regulate the effects such as adenylate cyclase activity, is the index components of Ixeris Sonchifolia Hance injection quality control.Therefore, in order to control product quality, to the nucleoside compound in Ixeris Sonchifolia Hance injection set up quick, easy, assay method has important practical significance accurately.
At present, the nucleoside compound content assaying method mainly contains ultraviolet spectrophotometry, thin-layered chromatography, high efficiency liquid phase-mass spectroscopy, capillary electrophoresis etc.Ultraviolet method and thin-layer method specificity are relatively poor, and liquid-matter and capillary electrophoresis are had relatively high expectations to instrument.
Summary of the invention
The objective of the invention is to provide for above-mentioned deficiency a kind of employing HPLC-DAD technology to set up the analytical approach of measuring simultaneously 10 kinds of nucleoside compounds (5 kinds of nucleosides: uridine, adenosine, guanosine, cytidine, thymidine and 5 kinds of bases: uracil, adenine, guanine, cytimidine, thymine) component content, and application the method is measured the content of main nucleoside compound in Ixeris Sonchifolia Hance injection, to for the further research of parenteral solution water soluble ingredient lays the foundation, be the reference frame of its quality control provider science of law simultaneously.
Technical solution of the present invention is: the HPLC-DAD method is measured the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection simultaneously, and its step is as follows:
(1) mix the preparation of reference substance solution:
It is appropriate that precision takes cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine reference substance respectively, adds water and make and contain cytimidine 0.50 μ gmL
-1, uracil 1.20 μ gmL
-1, cytidine 17.50 μ gmL
-1, guanine 20.00 μ gmL
-1, uridine 40.00 μ gmL
-1, thymine 2.50 μ gmL
-1, adenine 10.00 μ gmL
-1, guanosine 50.00 μ gmL
-1, thymidine 10.00 μ gmL
-1, adenosine 20.00 μ gmL
-1The mixing reference substance solution, ultrasonic dissolution shakes up, and namely gets to mix the reference substance storing solution.
(2) preparation of need testing solution:
Get Ixeris Sonchifolia Hance injection (medicine finished product), adopt 0. 45 μ m filtering with microporous membrane, as need testing solution, to be measured.
(3) determination method: adopt HPLC-DAD method accurate mixing reference substance solution and the need testing solution drawn respectively, Injection Detector is measured, and be get final product.
Chromatographic condition: chromatographic column: Diamonsil C
18(2); Detect wavelength: 260 nm; Flow velocity: 1 mLmin
-1Column temperature: 35 ℃; Sample size: 10 μ L; Condition of gradient elution: 0-5min, volume fraction is 2% acetonitrile isocratic elution, and the 5-9min linearity increases to 9% acetonitrile, and the 9-15min linearity increases to 11% acetonitrile, and the 15-25min linearity increases to 40% acetonitrile.
Above-mentioned steps (3) determination method comprises linear relationship mensuration; Namely accurate the absorption mixed reference substance storing solution 5 mL and is placed in 10 mL measuring bottles respectively, is diluted with water to scale, namely gets cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine and is respectively 0.25 μ gmL
-1, 0.60 μ gmL
-1, 8.75 μ gmL
-1, 10.00 μ gmL
-1, 20.00 μ gmL
-1, 1.25 μ gmL
-1, 5.00 μ gmL
-1, 25.00 μ gmL
-1, 5.00 μ gmL
-1, 10.00 μ gmL
-1Mixed solution, be diluted with water to successively scale, get series standard solution, HPLC measures.
Above-mentioned steps (3) determination method comprises precision mensuration; Namely precision is drawn hybrid standard product solution: the concentration of cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine is respectively 0.125,0.300,4.375,5.000,10.00,0.625,2.500,12.50,2.500,5.000 μ gmL
-1Mixed solution, under chromatographic condition, continuous sample introduction 6 times records peak area.
Above-mentioned steps (3) determination method comprises repeated experiment; Namely get Ixeris Sonchifolia Hance injection, test solution is made in parallel laboratory test 6 times, under chromatographic condition, measures respectively 6 times, calculates the chromatographic peak area of every kind of reference substance.
Above-mentioned steps (3) determination method comprises stability experiment; Namely get Ixeris Sonchifolia Hance injection, room temperature is placed, and in 0,2,4,6,8,12 h sample introductions, under chromatographic condition, peak area is recorded in the sample introduction analysis.
Above-mentioned steps (3) determination method comprises recovery experiment; Be that precision measures Ixeris Sonchifolia Hance injection 5.0 mL, parallel 6 parts, be placed in respectively 10 mL measuring bottles, precision adds and mixes reference substance solution 5.0 mL, wherein cytimidine 0.14 μ gmL respectively
-1, uracil 0.27 μ gmL
-1, cytidine 3.10 μ gmL
-1, guanine 4.60 μ gmL
-1, uridine 6.00 μ gmL
-1, thymine 1.20 μ gmL
-1, adenine 2.90 μ gmL
-1, guanosine 7.2 μ gmL
-1, thymidine 1.60 μ gmL
-1, adenosine 5.00 μ gmL
-1Shake up, under chromatographic condition, peak area is recorded in the sample introduction analysis, calculates average recovery.
advantage of the present invention is: 1, experimental result shows, gradient elution is better at the separating effect of acetonitrile-water gradient elution system, and the HPLC-DAD method is easy and simple to handle, gradient elution can obtain desirable separating effect under suitable chromatographic condition, under the chromatographic condition that the present invention selectes, both can complete analysis in the chromatographic peak 15min of nucleosides material in Ixeris Sonchifolia Hance injection, good separation, this method is measured fast, result is accurate, have good stability, it is a kind of reliable method of measuring the Ixeris Sonchifolia Hance injection Nucleosides, can be Ixeris Sonchifolia Hance injection quality evaluation system experimental basis is provided.2, by the analysis to 18 batches of Ixeris Sonchifolia Hance injection Nucleosides, result shows, contain 9 kinds of nucleosides in this parenteral solution, wherein content is higher guanosine, uridine, adenosine, guanine and cytidine etc., in the different batches parenteral solution, each nucleosides content has certain difference, but the adenosine differences between batches relevant to clinical efficacy are less.The injection production technology of explanation take nucleosides as index is comparatively stable, and also other gradient elutions of prompting reply are carried out correlative study, more comprehensively estimate to quality and security to Ixeris Sonchifolia Hance injection.3, the present invention sets up that the analytical approach of measuring simultaneously 10 kinds of gradient elutions in Ixeris Sonchifolia Hance injection is simple, good reproducibility, accurately and reliably, can be the Ixeris Sonchifolia Hance injection quality control foundation is provided.
Below in conjunction with accompanying drawing, embodiment, embodiments of the present invention are described in further detail.
Description of drawings
Fig. 1 is that the present invention mixes reference substance solution HPLC spectrogram.
Fig. 2 is need testing solution HPLC spectrogram of the present invention.
Embodiment
Embodiment
The HPLC-DAD method is measured the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection simultaneously:
1 instrument and reagent
1.1 instrument
The Waters-e2695-HPLC system (comprises the quaternary gradient pump, online vacuum degassing machine, automatic sampler, column oven, 2998 photodiode array detector PDA, Empower 2 chromatographic work stations), Sartorius BT125D type analysis balance (Sai Duolisi scientific instrument (Beijing) company limited), KH 2200B type ultrasonic cleaner (Kunshan standing grain wound ultrasonic instrument company limited)
1.2 reagent
Cytimidine reference substance (lot number: C94998BGF0, Tianjin Skien think biochemical technology company limited); Uracil reference substance (lot number: 100469-200401, Nat'l Pharmaceutical ﹠ Biological Products Control Institute); Cytidine reference substance (lot number: 101056874, SIGMA company); Guanine reference substance (lot number: HCCTH-BG, Shanghai changes into industrial development company limited); Uridine reference substance (lot number: 1001290893, SIGMA company); Thymine reference substance (lot number: T55620BGF0, Tianjin Skien think biochemical technology company limited); Adenine reference substance (lot number: A73241BGF0, Tianjin Skien think biochemical technology company limited); Guanosine reference substance (lot number: G24210BGQ0, Tianjin Skien think biochemical technology company limited); Thymidine reference substance (lot number: T55590BGF0, Tianjin Skien think biochemical technology company limited); Adenosine reference substance (lot number: 110879-200202, Nat'l Pharmaceutical ﹠ Biological Products Control Institute), all reference substance purity are calculated greater than 99% through the HPLC areas of peak normalization method.
Ixeris Sonchifolia Hance injection (Tonghua Huaxia Pharmaceutical Co., Ltd. provides, lot number: 120311T, 120312T, 120314T, 120315T, 120316T, 120317T, 129318T, 120319T, 120312,120532,120109,110601,111011,120438,120625,120209,111105,111218).Acetonitrile (chromatographically pure, SIGMA company), methyl alcohol (chromatographically pure, Concord, Tianjin Science and Technology Ltd.), ultrapure water, other reagent be analyze pure.
2 methods and result
2.1 mix the preparation of reference substance solution
It is appropriate that precision takes cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine reference substance respectively, adds water and make and contain cytimidine 0.50 μ gmL
-1, uracil 1.20 μ gmL
-1, cytidine 17.50 μ gmL
-1, guanine 20.00 μ gmL
-1, uridine 40.00 μ gmL
-1, thymine 2.50 μ gmL
-1, adenine 10.00 μ gmL
-1, guanosine 50.00 μ gmL
-1, thymidine 10.00 μ gmL
-1, adenosine 20.00 μ gmL
-1The mixing reference substance solution, ultrasonic dissolution shakes up, and namely gets to mix the reference substance storing solution.
2.2 the preparation of need testing solution
Get Ixeris Sonchifolia Hance injection, adopt 0. 45 μ m filtering with microporous membrane, as need testing solution, to be measured.
2.3 chromatographic condition
Chromatographic column: Diamonsil C
18(2) (200 mm * 4.6 mm, 5 μ m); Detect wavelength: 260 nm; Flow velocity: 1 mLmin
-1Column temperature: 35 ℃; Sample size: 10 μ L; Condition of gradient elution: 0-5min, volume fraction is 2% acetonitrile isocratic elution, and the 5-9min linearity increases to 9% acetonitrile, and the 9-15min linearity increases to 11% acetonitrile, and the 15-25min linearity increases to 40% acetonitrile.
2.4 specificity experiment
Under " 2.3 " chromatographic condition, cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, the mixing reference substance solution of adenosine and the HPLC spectrogram of Ixeris Sonchifolia Hance injection need testing solution are seen Fig. 1,2, in mixing reference substance and Ixeris Sonchifolia Hance injection test sample, 10 kinds of gradient elutions reach baseline separation.Referring to Fig. 1,2,1 cytimidines, 2 uracils, 3 cytidines, 4 guanines, 5 uridines, 6 thymines, 7 adenines, 8 guanosines, 9 thymidines, 10 adenosines.
2.5 linear relationship is investigated
(cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine are respectively 0.50 μ gmL to accurate absorption mixing reference substance storing solution respectively
-1, 1.20 μ gmL
-1, 17.50 μ gmL
-1, 20.00 μ gmL
-1, 40.00 μ gmL
-1, 2.50 μ gmL
-1, 10.00 μ gmL
-1, 50.00 μ gmL
-1, 10.00 μ gmL
-1, 20.00 μ gmL
-1Mixed solution) 5 mL are placed in 10 mL measuring bottles, are diluted with water to scale, namely get cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine and be respectively 0.25 μ gmL
-1, 0.60 μ gmL
-1, 8.75 μ gmL
-1, 10.00 μ gmL
-1, 20.00 μ gmL
-1, 1.25 μ gmL
-1, 5.00 μ gmL
-1, 25.00 μ gmL
-1, 5.00 μ gmL
-1, 10.00 μ gmL
-1Mixed solution, be diluted with water to successively scale, get series standard solution, HPLC measures.Take peak area (Y) as ordinate, reference substance concentration (X) is horizontal ordinate, carries out linear regression, draws regression equation and the related coefficient of each compound, the results are shown in Table 1.Result shows, 10 kinds of gradient elutions are good in range of linearity internal linear relation.
Regression equation, related coefficient and the range of linearity of 10 kinds of gradient elutions of table 1
| Compound | Regression equation | Coefficient R | The range of linearity (μ gmL -1) |
| Cytimidine | Y= 3.35×10 4X + 4.09×10 2 | 0.9995 | 0.02-0.50 |
| Uracil | Y = 2.20×10 4X +1.16×10 3 | 0.9995 | 0.04-1.20 |
| Cytidine | Y= 1.89×10 4X + 2.04×10 3 | 0.9995 | 0.55-17.50 |
| Guanine | Y = 6.92×10 4X +2.80×10 2 | 0.9990 | 0.63-20.00 |
| Uridine | Y = 2.47×10 4X -4.25×10 3 | 0. 9995 | 1.25-20.00 |
| Thymine | Y= 3.04×10 4X + 8.18×10 2 | 0.9990 | 0.08-2.50 |
| Adenine | Y= 5.46×10 4X +7.27×10 3 | 0.9995 | 0.31-10.00 |
| Guanosine | Y = 2.01×10 4X -7.73×10 3 | 0.9995 | 1.56-50.00 |
| Thymidine | Y= 2.06×10 4X- 1.22×10 3 | 0.9995 | 0.31-10.00 |
| Adenosine | Y= 3.21×10 4X-1.29×10 4 | 0.9995 | 0.63-20.00 |
2.6 Precision Experiment
The accurate hybrid standard product solution of drawing: the concentration of cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine is respectively 0.125,0.300,4.375,5.000,10.00,0.625,2.500,12.50,2.500,5.000 μ gmL
-1Mixed solution, under " 2.3 " chromatographic condition, continuous sample introduction 6 times records peak area.The withinday precision RSD (n=6) of cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine is respectively 1.5%, 1.7%, 0.3%, 0.1%, 0.1%, 0.5%, 0.2%, 0.2%, 0.2%, 0.2% as a result.Result shows, instrument precision is good.
2.7 repeated experiment
(lot number: 120319T), parallel laboratory test 6 times is made test solution by " 2.2 " below method, under " 2.3 " chromatographic condition, measures respectively (n=6), calculates the chromatographic peak area of every kind of reference substance to get Ixeris Sonchifolia Hance injection.As a result, the average content of cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine is 0.763,0.131,2.831,0.438,6.138,1.911,7.123,0.903,4.598 μ gmL
-1RSD is respectively 3.0%, 4.2%, 2.3%, 4.3%, 1.8%, 3.7%, 0.8%, 3.1% and 2. 2%, and result shows, the repeatability of the method is good.
2.8 stability experiment
(lot number: 120319T), room temperature is placed, and in 0,2,4,6,8,12 h sample introductions, under " 2.3 " chromatographic condition, peak area is recorded in the sample introduction analysis to get Ixeris Sonchifolia Hance injection.Cytimidine as a result, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, the RSD of adenosine peak area is respectively 3.2%, 2.8%, 3.4%, 3.9%, 1.9%, 3.3%, 2.1%, 2.0% and 2.9%.Result shows, need testing solution is stable in 12 h.
2.9 recovery experiment
Precision measures the Ixeris Sonchifolia Hance injection of known content, and (lot number: 120319T) 5.0 mL, are placed in respectively 10 mL measuring bottles, respectively accurate mixing reference substance solution 5.0 mL that add certain mass concentration, wherein cytimidine 0.14 μ gmL by parallel 6 parts
-1, uracil 0.27 μ gmL
-1, cytidine 3.10 μ gmL
-1, guanine 4.60 μ gmL
-1, uridine 6.00 μ gmL
-1, thymine 1.20 μ gmL
-1, adenine 2.90 μ gmL
-1, guanosine 7.2 μ gmL
-1, thymidine 1.60 μ gmL
-1, adenosine 5.00 μ gmL
-1Shake up, under " 2.3 " chromatographic condition, peak area is recorded in the sample introduction analysis, calculates average recovery, the results are shown in Table 2.Result shows, average recovery rate is 98.63% ~ 102.96%, RSD≤2.61%.
The recovery (n=6) of 10 kinds of gradient elutions of table 2
| Compound | The content of sample (μ g) | Reference substance content (μ g) | Practical measurement amount (μ g) | The recovery (%) | RSD(%) |
| Cytimidine | 0.62 | 0.68 | 1.30 | 99.61 | 2.55 |
| Uracil | 1.32 | 1.35 | 2.69 | 101.08 | 2.10 |
| Cytidine | 14.50 | 15.50 | 30.24 | 101.54 | 2.61 |
| Guanine | 23.00 | 23.00 | 46.22 | 100.95 | 2.33 |
| Uridine | 32.00 | 30.00 | 62.46 | 101.53 | 2.57 |
| Thymine | 0.00 | 6.00 | 6.18 | 102.92 | 2.27 |
| Adenine | 11.00 | 14.50 | 25.38 | 99.12 | 1.98 |
| Guanosine | 33.50 | 36.00 | 70.27 | 102.14 | 2.27 |
| Thymidine | 8.38 | 8.00 | 16.27 | 98.63 | 2.54 |
| Adenosine | 25.80 | 25.00 | 51.54 | 102.96 | 0.69 |
2.10 sample size is measured
Get Ixeris Sonchifolia Hance injection appropriate, by " a 2.2 " below legal system available examination solution, under " 2.3 " chromatographic condition, peak area is recorded in the sample introduction analysis, and with the content of regression equation calculation nucleoside compound, measurement result sees Table 3.Result shows, comprises other the 9 kinds of gradient elutions except thymine in Ixeris Sonchifolia Hance injection.
Table 3 different batches Ixeris Sonchifolia Hance injection Nucleosides content (μ gmL
-1
)
| Lot number | Cytimidine | Uracil | Cytidine | Guanine | Uridine | Adenine | Guanosine | Thymidine | Adenosine | Summation |
| 120311T | 0.09 | 0.26 | 2.78 | 4.94 | 6.78 | 3.47 | 8.05 | 1.01 | 5.05 | 32.43 |
| 120312T | 0.11 | 0.25 | 2.74 | 6.95 | 6.28 | 2.33 | 7.65 | 0.74 | 4.39 | 31.44 |
| 120314T | 0.08 | 0.23 | 2.57 | 4.47 | 6.11 | 2.41 | 7.65 | 0.75 | 5.42 | 29.69 |
| 120315T | 0.11 | 0.30 | 2.56 | 4.83 | 6.43 | 2.44 | 9.16 | 1.14 | 4.90 | 31.87 |
| 120316T | 0.11 | 0.22 | 2.91 | 5.06 | 7.38 | 2.90 | 8.95 | 1.34 | 4.87 | 33.74 |
| 120317T | 0.09 | 0.29 | 2.76 | 6.69 | 5.53 | 2.55 | 7.49 | 0.84 | 4.26 | 30.50 |
| 129318T | 0.11 | 0.34 | 2.72 | 4.66 | 6.77 | 2.34 | 7.64 | 1.64 | 4.87 | 31.09 |
| 120319T | 0.10 | 0.24 | 3.32 | 4.93 | 6.34 | 2.42 | 7.30 | 1.00 | 4.46 | 30.11 |
| 120312 | 0.07 | 1.92 | 6.62 | 0.20 | 4.66 | 1.28 | 5.21 | 0.80 | 4.34 | 25.10 |
| 120532 | 0.12 | 2.69 | 5.86 | 0.41 | 6.10 | 2.05 | 7.18 | 1.15 | 3.87 | 29.43 |
| 120109 | 0.11 | 2.11 | 12.20 | 0.25 | 5.57 | 1.55 | 6.91 | 0.89 | 5.51 | 35.09 |
| 110601 | 0.08 | 1.76 | 7.75 | 0.22 | 4.90 | 1.38 | 6.53 | 0.86 | 4.28 | 27.76 |
| 111011 | 0.07 | 1.73 | 6.15 | 0.21 | 4.42 | 1.04 | 5.73 | 0.73 | 4.06 | 24.16 |
| 120438 | 0.10 | 2.40 | 6.23 | 0.40 | 5.31 | 1.96 | 7.26 | 0.95 | 4.33 | 28.93 |
| 120625 | 0.10 | 1.92 | 12.51 | 0.30 | 4.68 | 1.34 | 6.31 | 0.91 | 4.89 | 32.96 |
| 120209 | 0.09 | 1.89 | 11.68 | 0.27 | 5.35 | 1.69 | 6.49 | 0.92 | 5.30 | 33.69 |
| 111105 | 0.09 | 1.71 | 10.08 | 0.16 | 4.82 | 1.18 | 5.55 | 0.84 | 4.32 | 28.75 |
| 111218 | 0.06 | 1.75 | 9.89 | 0.18 | 4.43 | 1.14 | 5.38 | 0.76 | 3.97 | 27.55 |
| C | 0.09 | 1.22 | 6.18 | 2.51 | 5.66 | 1.97 | 7.02 | 0.96 | 4.62 | 30.24 |
| RSD(%) | 17.8 | 74.5 | 59.5 | 105.9 | 15.9 | 34.7 | 16.1 | 24.4 | 10.8 | 9.7 |
3 discuss
Adopt the DAD detecting device to carry out full wavelength scanner (200 ~ 400 nm), analyze the ultraviolet spectrum of 10 kinds of nucleoside compounds, result shows, when detecting wavelength and being 260 nm, 10 kinds of compounds all have more by force and absorb, and good separation.Consider response and the disturbing factor of each chromatographic peak when detecting, finally select 260nm to detect wavelength.
Gradient elution is alkalescent, and polarity is large, more difficult separation.Respectively methanol-water and acetonitrile-water are investigated as flow phase system in experiment.Result shows, gradient elution is better at the separating effect of acetonitrile-water gradient elution system, and the HPLC-DAD method is easy and simple to handle, gradient elution can obtain desirable separating effect under suitable chromatographic condition, under the chromatographic condition that this paper selectes, both can complete analysis in the chromatographic peak 15min of nucleosides material in Ixeris Sonchifolia Hance injection, good separation, this method is measured fast, result is accurate, have good stability, be a kind of reliable method of measuring the Ixeris Sonchifolia Hance injection Nucleosides, can be Ixeris Sonchifolia Hance injection quality evaluation system experimental basis is provided.
4 conclusions
By the analysis to 18 batches of Ixeris Sonchifolia Hance injection Nucleosides, result shows, contain 9 kinds of nucleosides in this parenteral solution, wherein content is higher guanosine, uridine, adenosine, guanine and cytidine etc., in the different batches parenteral solution, each nucleosides content has certain difference, but the adenosine differences between batches relevant to clinical efficacy are less.The injection production technology of explanation take nucleosides as index is comparatively stable, and also other gradient elutions of prompting reply are carried out correlative study, more comprehensively estimate to quality and security to Ixeris Sonchifolia Hance injection.
Claims (6)
1. a HPLC-DAD method is measured the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection simultaneously, it is characterized in that step is as follows:
(1) mix the preparation of reference substance solution:
It is appropriate that precision takes cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine reference substance respectively, adds water and make and contain cytimidine 0.50 μ gmL
-1, uracil 1.20 μ gmL
-1, cytidine 17.50 μ gmL
-1, guanine 20.00 μ gmL
-1, uridine 40.00 μ gmL
-1, thymine 2.50 μ gmL
-1, adenine 10.00 μ gmL
-1, guanosine 50.00 μ gmL
-1, thymidine 10.00 μ gmL
-1, adenosine 20.00 μ gmL
-1The mixing reference substance solution, ultrasonic dissolution shakes up, and namely gets to mix the reference substance storing solution;
(2) preparation of need testing solution:
Get Ixeris Sonchifolia Hance injection, adopt 0. 45 μ m filtering with microporous membrane, as need testing solution, to be measured;
(3) determination method: adopt HPLC-DAD method accurate mixing reference substance solution and the need testing solution drawn respectively, Injection Detector is measured, and be get final product;
Chromatographic condition: chromatographic column: Diamonsil C
18(2); Detect wavelength: 260 nm; Flow velocity: 1 mLmin
-1Column temperature: 35 ℃; Sample size: 10 μ L; Condition of gradient elution: 0-5min, 2% acetonitrile isocratic elution, the 5-9min linearity increases to 9% acetonitrile, and the 9-15min linearity increases to 11% acetonitrile, and the 15-25min linearity increases to 40% acetonitrile.
2. measure simultaneously the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection according to HPLC-DAD method claimed in claim 1, it is characterized in that step (3) determination method comprises linear relationship mensuration; Namely accurate the absorption mixed reference substance storing solution 5 mL and is placed in 10 mL measuring bottles respectively, is diluted with water to scale, namely gets cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine and is respectively 0.25 μ gmL
-1, 0.60 μ gmL
-1, 8.75 μ gmL
-1, 10.00 μ gmL
-1, 20.00 μ gmL
-1, 1.25 μ gmL
-1, 5.00 μ gmL
-1, 25.00 μ gmL
-1, 5.00 μ gmL
-1, 10.00 μ gmL
-1Mixed solution, be diluted with water to successively scale, get series standard solution, HPLC measures.
3. measure simultaneously the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection according to HPLC-DAD method claimed in claim 1, it is characterized in that step (3) determination method comprises precision mensuration; Namely precision is drawn hybrid standard product solution: the concentration of cytimidine, uracil, cytidine, guanine, uridine, thymine, adenine, guanosine, thymidine, adenosine is respectively 0.125,0.300,4.375,5.000,10.00,0.625,2.500,12.50,2.500,5.000 μ gmL
-1Mixed solution, under chromatographic condition, continuous sample introduction 6 times records peak area.
4. measure simultaneously the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection according to HPLC-DAD method claimed in claim 1, it is characterized in that step (3) determination method comprises repeated experiment; Namely get Ixeris Sonchifolia Hance injection, test solution is made in parallel laboratory test 6 times, under chromatographic condition, measures respectively 6 times, calculates the chromatographic peak area of every kind of reference substance.
5. measure simultaneously the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection according to HPLC-DAD method claimed in claim 1, it is characterized in that step (3) determination method comprises stability experiment; Namely get Ixeris Sonchifolia Hance injection, room temperature is placed, and in 0,2,4,6,8,12 h sample introductions, under chromatographic condition, peak area is recorded in the sample introduction analysis.
6. measure simultaneously the method for 10 kinds of gradient elution content in Ixeris Sonchifolia Hance injection according to HPLC-DAD method claimed in claim 1, it is characterized in that step (3) determination method comprises recovery experiment; Be that precision measures Ixeris Sonchifolia Hance injection 5.0 mL, parallel 6 parts, be placed in respectively 10 mL measuring bottles, precision adds and mixes reference substance solution 5.0 mL, wherein cytimidine 0.14 μ gmL respectively
-1, uracil 0.27 μ gmL
-1, cytidine 3.10 μ gmL
-1, guanine 4.60 μ gmL
-1, uridine 6.00 μ gmL
-1, thymine 1.20 μ gmL
-1, adenine 2.90 μ gmL
-1, guanosine 7.2 μ gmL
-1, thymidine 1.60 μ gmL
-1, adenosine 5.00 μ gmL
-1Shake up, under chromatographic condition, peak area is recorded in the sample introduction analysis, calculates average recovery.
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| CN104502511A (en) * | 2015-01-04 | 2015-04-08 | 西南民族大学 | Detection method for four nucleoside ingredients in allium wallichii kunth |
| CN104502511B (en) * | 2015-01-04 | 2016-08-17 | 西南民族大学 | The detection method of four kinds of gradient elutions in Allium wallichii |
| CN109959732A (en) * | 2017-12-26 | 2019-07-02 | 内蒙古京新药业有限公司 | The separation method of Kangfuxin Liquid finger-print and its effective constituents A |
| CN108333280A (en) * | 2018-01-26 | 2018-07-27 | 上海上药第生化药业有限公司 | The separation method of nucleosides material and its application in a kind of Pericarpium Trichosanthis injection |
| CN108709949A (en) * | 2018-05-29 | 2018-10-26 | 江西国药有限责任公司 | A kind of detection method of the fermentation cordyceps Cs-4 prepared slices of Chinese crude drugs |
| CN110887905A (en) * | 2018-11-22 | 2020-03-17 | 江西诚志生物工程有限公司 | Method for detecting key intermediate β -thymidine of anti-HIV drug |
| JP2020085623A (en) * | 2018-11-22 | 2020-06-04 | フォーデイズ株式会社 | Method for quantifying nucleotide, nucleoside and/or base derived from nucleic acid (DNA and RNA) |
| CN109580858A (en) * | 2019-01-31 | 2019-04-05 | 中山大学 | The extraction of 5 kinds of gradient elutions and its content assaying method in a kind of wide dragon |
| CN109932441A (en) * | 2019-03-01 | 2019-06-25 | 贵州瑞和制药有限公司 | A kind of method for building up of easypro liver injection for curing HPLC finger-print |
| CN109828060A (en) * | 2019-04-04 | 2019-05-31 | 陕西中医药大学 | The detection method of motherwort Nucleosides |
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