CN109884216A - A method for the determination of farnesal content in drug-loaded micelles by high performance liquid chromatography - Google Patents
A method for the determination of farnesal content in drug-loaded micelles by high performance liquid chromatography Download PDFInfo
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Abstract
本发明涉及一种高效液相色谱测定载药胶束中法尼醛含量的方法，属于药物分析领域。采用安捷伦‑1260高效液相色谱仪，采用紫外检测器，检测波长为216nm；色谱条件：色谱柱选用十八烷基硅烷键合硅胶为填充物的反向色谱柱；流动相优选为体积比为80：20的乙腈与水的混合物；流速为0.8～1.2mL/min，进样量为5～15μL，柱温为20～40℃。本发明方法具有操作简便，专属性强，准确度高，精密度良好等特点，日内和日间标准偏差均不超过1.2％，因此本发明方法能够准确有效达对法尼醛进行含量测定的要求，能够准确地用于法尼醛负载的载药胶束的包封率、载药量、体外释放等实验中法尼醛含量的测试。
The invention relates to a method for measuring farnesal content in drug-loaded micelles by high performance liquid chromatography, and belongs to the field of drug analysis. Using Agilent-1260 high performance liquid chromatograph, using UV detector, the detection wavelength is 216nm; chromatographic conditions: the chromatographic column is a reverse chromatographic column with octadecylsilane-bonded silica gel as the filler; the mobile phase is preferably a volume ratio of 80:20 mixture of acetonitrile and water; the flow rate is 0.8-1.2 mL/min, the injection volume is 5-15 μL, and the column temperature is 20-40°C. The method of the invention has the characteristics of simple operation, strong specificity, high accuracy, good precision and the like, and the intra-day and inter-day standard deviations do not exceed 1.2%, so the method of the invention can accurately and effectively meet the requirements for the content determination of farnesal , which can be accurately used to test the farnesal content in experiments such as the encapsulation efficiency, drug loading, and in vitro release of farnesal-loaded drug-loaded micelles.
Description
Technical field
The invention belongs to Pharmaceutical Analysis fields, it is more particularly related to which a kind of measurement of high performance liquid chromatography carries medicine
The method of method Buddhist nun aldehyde in micella.
Background technique
Method Buddhist nun aldehyde (Farnesal, Far), also known as farnesal, belong to sequiterpene olefine aldehydr, mainly from lemon grass (Cymbopogon citratus) and horse
In whip grass, chemical name are as follows: 3,7,11- trimethyls -2,6, three olefine aldehydr of 10- dodecane, molecular formula C15H24O, molecular weight are
220.35.The water solubility of method Buddhist nun's aldehyde is very poor, is practically insoluble in water, this product can be aoxidized by farnesol (Farnesal) or dehydrogenation system
?.Research shows that method Buddhist nun's aldehyde is non-toxic in vivo, and can inhibit the growth of various bacteria microorganism, as staphylococcus aureus,
Staphylococcus epidermis and Candida albicans etc..In addition method Buddhist nun aldehyde can also influence the permeability of streptococcus mutans biomembrane, reduce
The glycolysis of Streptococcus mutans；It can inhibit bacterium and generate polysaccharide, acidic metabolite, destroy the acid resistance of Streptococcus mutans, grind
Study carefully and shows that method Buddhist nun aldehyde just has good inhibitory effect to Streptococcus mutans in low concentration.
Method Buddhist nun aldehyde structure is as shown in following formula one:
Through retrieving, there are no a kind of bodies simple, efficient and specificity is strong, accuracy is high, reproducible in the prior art
The method of outer measuring method Buddhist nun aldehyde.
Based on the above reasons, the application is proposed.
Summary of the invention
In view of the deficiencies in the prior art, it is measured the purpose of the present invention is to provide a kind of high performance liquid chromatography and carries medicine
The method of method Buddhist nun aldehyde in micella, this method have easy to operate, and specificity is strong, and accuracy is high, and precision, repeatability are good
The features such as, it can effectively reach the requirement that assay is carried out to method Buddhist nun's aldehyde.
To achieve the above objectives, the technical solution adopted by the present invention is as follows:
A kind of method that high performance liquid chromatography measures method Buddhist nun's aldehyde in carrier micelle, using the efficient liquid of Agilent -1260
Chromatography, using UV detector, Detection wavelength 216nm includes the following steps:
(1) chromatographic condition: it is the reverse chromatograms column of filler that chromatographic column, which selects octadecylsilane chemically bonded silica,；Mobile phase
For the mixture or methanol of acetonitrile and water and the mixture of water or acetonitrile and any in the mixture of 0.01% phosphoric acid solution
Kind；Wherein: the volume ratio of acetonitrile and water is 60~90:10~40, and the volume ratio of methanol and water is 60~90:10~40, acetonitrile
Volume ratio with 0.01% phosphoric acid solution is 60~90:10~40；Flow velocity is 0.8~1.2mL/min, and sample volume is 5~15 μ
L, column temperature are 20~40 DEG C；
(2) Specification Curve of Increasing: precision weighing method Buddhist nun aldehyde (reference substance) in right amount, is placed in volumetric flask, with flowing phased soln,
And it is diluted to scale, as method Buddhist nun's aldehyde standard solution stock solution, the standard solution stock solution is then diluted to method Buddhist nun step by step
Aldehyde is the solution of 10,25,50,100,150,200,250,300,350,400 μ g/mL, presses step (1) described chromatography respectively
Condition measurement, calculates separately the peak area under same retention time, using content x as abscissa, using peak area y as ordinate, does
Calibration curve equation y=f (x) out, content are 10~400 μ g/mL；
(3) sample to be tested is prepared: precision weighs that sample to be tested is appropriate, adds mobile phase dissolved dilution at the solution of debita spissitudo
Afterwards, it filters；
(4) it detects the content of method Buddhist nun's aldehyde in method Buddhist nun aldehyde sample to be measured: method Buddhist nun aldehyde sample solution to be measured is pressed into step (1) respectively
The chromatographic condition test, calculates the peak area under same retention time, brings step (2) resulting calibration curve equation into, count
The concentration for obtaining method Buddhist nun aldehyde sample solution to be measured is calculated, then calculates the content of method Buddhist nun aldehyde in sample by the sampling amount of sample.
Further, above-mentioned technical proposal, the chromatographic column select Agilent C18 column 4.6*150mm, 5 μm,
Waters C18 column 4.6*150mm, 5 μm or Shimadzu C18 column 4.6*150mm, one of 5 μm, it is preferred to use
Agilent C18 column 4.6*150mm, 5 μm.
Further, above-mentioned technical proposal, step (1) described mobile phase are preferably the mixture of acetonitrile and water, the second
The volume ratio of nitrile and water is 80:20.
Further, above-mentioned technical proposal, step (1) described flow velocity is preferably 1.0mL/min.
Further, above-mentioned technical proposal, step (1) described column temperature is preferably 25 DEG C.
Further, above-mentioned technical proposal, step (1) sample volume are 10 μ L.
Further, above-mentioned technical proposal, step (1) filtering preferably use 0.22 μm of miillpore filter.
Compared with prior art, method Buddhist nun's aldehyde in a kind of high performance liquid chromatography measurement carrier micelle of the present invention
The advantages of method, is:
(1) the features such as the method for the present invention has easy to operate, and specificity is strong, and accuracy is high, and precision is good, in a few days and day
Between standard deviation be no more than 1.2%, therefore the method for the present invention can accurate and effective reach and assay is carried out to method Buddhist nun's aldehyde want
It asks, method Buddhist nun's aldehyde in the experiments such as encapsulation rate, drugloading rate, the release in vitro of carrier micelle of method Buddhist nun's aldehyde load can be accurately used for and contained
The test of amount.
(2) sample preparation that the method for the present invention is related to is convenient, easy to operate, and the method for the present invention is reproducible, and sample is flat
The RSD of row test is no more than in 1.5%.
(3) rate of recovery of the present invention is high, and average recovery rate is between 98.0%~102%, therefore test method of the present invention is quasi-
Exactness is high.
Detailed description of the invention
Fig. 1 is the canonical plotting of method Buddhist nun's aldehyde in the embodiment of the present invention 1.
Fig. 2 is the liquid chromatogram that specificity investigates method Buddhist nun aldehyde (A), blank micella (B), carrier micelle (C) in experiment.
Specific embodiment
Below with reference to specific implementation case, invention is further described in detail.The implementation case is with the technology of the present invention
Premised under implemented, now provide detailed embodiment and specific operating process illustrate the present invention it is creative,
But protection scope of the present invention case study on implementation not limited to the following.
It is not intended to limit the scope of the invention for a better understanding of the present invention, expression dosage used in this application,
All numbers of percentage and other numerical value, are understood to be modified with word " about " in all cases.Therefore,
Unless stated otherwise, otherwise digital parameters listed in specification and appended book are all approximations, may
It can be changed according to the difference for the desirable properties for attempting to obtain.Each digital parameters at least should be considered as according to being reported
Effective digital and obtained by the conventional method of rounding up.
The content being not described in detail in this specification belongs to the prior art well known to professional and technical personnel in the field.
It should be noted that the following carrier micelles used in the examples of the present invention are PPi-Far-PMs, it is using following
Method is made:
(1) synthesis of compound 1 (Boc-PEG)
Take OH-PEG2000-NHNH2(500mg, 0.25mmol) is dissolved in proper amount of methanol, adds the triethylamine of 1 equivalent
(25.3mg, 0.25mmol) makes solution be cooled to 0 DEG C, is slowly added to di-tert-butyl dicarbonate (65.5mg, 0.30mmol), 0 DEG C
Lower stirring 1h, then it is slowly increased to room temperature, reaction is for 24 hours.End of reaction, vacuum rotary steam remove methanol, remaining a small amount of solvent, then with greatly
Measure anhydrous ether precipitating.Precipitating is taken to be dissolved with a small amount of distilled water, dialyse 12h, vacuum freeze-drying, -20 DEG C of preservations.Gained compound 1
Yield is 72.4%.
The characterization of compound 11H NMR,13C NMR characterization result is as follows:1H NMR(400MHz,Chloroform-d)δ
(ppm)7.01(s,1H),6.67(s,1H),4.27(s,2H),3.64(s,180H),1.45(s,9H)；13C NMR(101MHz,
Chloroform-d)δ(ppm)70.71,28.40.
(2) synthesis of compound 2 (Boc-PEG-PPi)
Compound 1 (500mg, 0.24mmol) and bromoacetic acid (43.4mg, 0.31mmol) is taken to be added to anhydrous dichloro jointly
In methane (DCM), solution is made to be cooled to 0 DEG C, is slowly added to 4-dimethylaminopyridine (DMAP, 2.9mg, 0.024mmol) and N,
N '-dicyclohexylcarbodiimide (DCC, 54.4mg, 0.26mmol), room temperature magnetic agitation react filtering for 24 hours, and decompression rotation is steamed
Organic solvent, remaining a small amount of solvent are sent out, then is precipitated with water ether.Filtering, precipitating are dissolved with a small amount of distilled water, are dialysed for 24 hours, vacuum
Freeze-drying, -20 DEG C of preservations.Three (tetrabutylammonium) hydrogen pyrophosphoric acids (TBAP, 415.1mg, 0.46mmol) are taken to be dissolved in appropriate anhydrous second
In nitrile, above-mentioned reaction product (500mg, 0.23mmol) is slowly added dropwise into solution.Room temperature magnetic agitation 12h, rotation have been evaporated
Solvent, in the residue obtained NaCl solution for being dissolved in suitable 25mM, and the 12h that dialyses in NaCl solution.Thick production after taking dialysis
Object is dialysed 12h in distilled water solution, to remove small molecule NaCl, the product of vacuum freeze-drying after purification, -20 DEG C of preservations.Gained
2 yield of compound is 80.7%.
The characterization of compound 21H NMR,13C NMR and31P NMR characterization result is as follows:1H NMR(400MHz,
Chloroform-d)δ(ppm)4.30-4.19(m,4H),3.87(s,1H),3.62(s,180H),2.45(s,2H),1.44(s,
9H)；13C NMR(101MHz,Chloroform-d)δ(ppm)155.82,70.67,69.36,68.88,65.47,64.93,
28.37；31P NMR(D2O,160MHz)δ(ppm)-9.49(m,2P).
(3) synthesis of compound 3 (PPi-PEG)
Compound 2 (500mg, 0.18mmol) is taken to be dissolved in the methylene chloride of trifluoroacetic acid (TFA, 41mg, 0.36mmol)
In solution, and 1h is stirred to react at 0 DEG C, revolving removes organic solvent, remaining a small amount of solvent, then is precipitated with water ether.Filtering is sunk
It forms sediment and is dissolved with a small amount of distilled water, dialyse 12h, vacuum freeze-drying, -20 DEG C of preservations.3 yield of gained compound is 80.2%.
The characterization of compound 31H NMR,13C NMR characterization result is as follows:1H NMR(400MHz,Chloroform-d)δ
(ppm)6.27(s,1H),4.27-4.21(m,4H),3.65(s,180H),1.87(s,3H)；13C NMR(101MHz,
Chloroform-d)δ(ppm)70.72,69.60,64.71.
(4) synthesis of compound 4 (PPi-PEG-hyd-Far)
(500mg, 0.19mmol) the He Fani aldehyde of compound 3 (61mg, 0.28mmol) is taken to be dissolved in proper amount of methanol, then to
A small amount of catalyst acetic acid is added in reaction solution.48h is reacted at room temperature, a large amount of cold anhydrous ethers are added into reaction solution keeps product heavy
Precipitation goes out, and decompression is filtered to doing, and gained precipitating is used ether repeated precipitation 3 times again after being dissolved with methylene chloride, and gained precipitates vacuum
It is dry, both obtain product.4 yield of gained compound is 79.8%.
The characterization of compound 41H NMR,13C NMR characterization result is as follows:1H NMR(400MHz,Chloroform-d)δ
(ppm)8.63(s,1H),7.80(m,1H),6.08(s,1H),5.07(m,2H),4.33(s,3H),3.63(s,182H),2.21
(s,2H),2.12(s,3H),2.03(s,2H),1.96(s,2H),1.85-1.82(m,2H),1.66(s,3H),1.57(s,
6H)；13C NMR(101MHz,Chloroform-d)δ(ppm)136.05,131.67,124.40,124.29,123.32,
123.13,72.80,70.70,70.40,69.48,40.20,39.84,26.84,26.21,25.90,17.89,17.47,
16.20.
(5) synthesis of compound 5 (PPi-Far-PMs)
Precision weighs 4mg method Buddhist nun's aldehyde, 20mg Far-PEG2000- PPi, 20mg mPEG2000-PLA2000The two is dissolved in
In 4mL acetonitrile, 55 DEG C are removed under reduced pressure solvent, obtain the mixed film of drug and carrier material；It measures 2mL UP water and round bottom is added
In flask, by bottle wall pharmaceutical film aquation；Ultrasonic 3min transfers to room temperature, and magnetic agitation 12h at room temperature；With 0.22 μ
The disposable injection filter filtering of m, the transparent and filtrate with light blue opalescence of gained is polypeptide drug-loaded micelle solution (PPi-Far-
PMs it), and in 4 DEG C stores, in case subsequent experimental uses.
In addition, the preparation method of the blank micella solution (Blank PMs) utilized in the following embodiments of the present invention with it is above-mentioned
The preparation method of the carrier micelle (PPi-Far-PMs) is essentially identical, and difference is only that: the preparation method of blank micella solution
In non-addition method Buddhist nun aldehyde, and by PEG2000Far-PEG in-PPi alternative embodiment 12000- PPi, other conditions are constant.
Embodiment 1
The method of method Buddhist nun's aldehyde in the high performance liquid chromatography measurement carrier micelle of the present embodiment, using Agilent -1260
High performance liquid chromatograph includes the following steps:
(1) selection of Detection wavelength
Precision weighs 5mg method Buddhist nun aldehyde (Farnesal, Far), is placed in the volumetric flask of 25mL, and acetonitrile is added to keep it completely molten
Solution, constant volume obtain method Buddhist nun's aldehyde stock solution (100 μ g/mL).Measurement method Buddhist nun's aldehyde stock solution is a small amount of, with dilution in acetonitrile to suitable concentration,
And using acetonitrile as blank control, it is scanned, is determined most in the wave-length coverage of 200-600nm using ultraviolet specrophotometer
Big absorbing wavelength.
By ultraviolet specrophotometer full wavelength scanner, select 216nm as UV Detection wavelength.
(2) chromatographic condition
This experiment uses the content of high effective liquid chromatography for measuring method Buddhist nun's aldehyde.Chromatographic column: Agilent ZORBAXSB-C18
(4.6×150mm,5μm).Mobile phase, acetonitrile: water=80:20 (v/v)；Detection wavelength, 216nm；Flow velocity, 1.0mL/min；Column
Temperature, 25 DEG C；Sample volume, 10 μ L.
(3) drafting of standard curve
Precision weighs 12.5mg method Buddhist nun's aldehyde, is placed in 25mL volumetric flask, graduation mark is dissolved and be settled to acetonitrile, obtain dense
Degree is method Buddhist nun's aldehyde stock solution of 500 μ g/mL, spare.It is accurate respectively again measure 0.1,0.25,0.5,1,1.5,2,2.5,3,3.5,
4 method Buddhist nun's aldehyde stock solution is placed in 5mL volumetric flask, with acetonitrile constant volume, be configured to 10,25,50,100,150,200,250,300,
350, various concentration method Buddhist nun's aldehyde solution of 400 μ g/mL.Method Buddhist nun's aldehyde respectively by step (2) chromatographic condition measurement series of concentrations is molten
The peak area of liquid, and be that ordinate carries out linear regression to concentration (C) with peak area (A), obtaining calibration curve equation is A=
15146C-29860, R2=0.9992 (n=3).Fig. 1 shows method Buddhist nun aldehyde concentration and peak within the scope of 10~400 μ g/mL of concentration
The linear relationship of area is good.
(4) sample to be tested is prepared: it is appropriate that precision weighs method Buddhist nun's aldehyde, adds methanol dissolve and is formulated into a certain concentration, use
0.22 μm of filtering with microporous membrane；
(5) content of method Buddhist nun's aldehyde in method Buddhist nun aldehyde sample to be measured is detected: by method Buddhist nun aldehyde sample solution to be measured by described in step (2)
Chromatographic condition test, calculates the peak area under same retention time, brings step (3) resulting calibration curve equation into, calculate
The concentration of method Buddhist nun aldehyde sample solution to be measured out, then calculate by the sampling amount of sample the content of method Buddhist nun aldehyde in sample.
Method Buddhist nun aldehyde (%)=method to be measured Buddhist nun's aldehyde sample solution concentration in sample/(method Buddhist nun aldehyde sampling weight * to be measured is dilute
Release the factor) * 100%, in which: dilution gfactor is the inverse of sample extension rate.
1.1 specificities are investigated
It is appropriate that precision weighs method Buddhist nun's aldehyde, adds methanol to dissolve and is formulated into a certain concentration, with 0.22 μm of filtering with microporous membrane,
By the content of method Buddhist nun's aldehyde in embodiment 1 step (2) content assaying method measurement filtrate, chromatogram is recorded；Prepare carrier micelle
And blank micella, accurate 500 μ L, the two kinds of micellar solutions that measure add methanol constant volume to graduation mark, ultrasound into 5mL volumetric flask respectively
It is demulsified after 5min, with 0.22 μm of filtering with microporous membrane, measures containing for method Buddhist nun's aldehyde in filtrate by step (2) content assaying method
Amount records chromatogram.
Specificity experimental result such as Fig. 2, in step (2) chromatographic condition, the retention time about 10.574min of method Buddhist nun's aldehyde,
Blank micella is no noiseless to the measurement of method Buddhist nun's aldehyde, shows that the chromatographic condition meets assay condition.
1.2 repeatability are investigated
The carrier micelle of the preparation method Buddhist nun's aldehyde is taken out 6 parts of isometric samples, respectively with dilution in acetonitrile to suitable concentration,
With 0.22 μm of filtering with microporous membrane, it is as shown in table 1 that method Buddhist nun's aldehyde peak area data are measured by chromatographic condition under step (2).Pass through meter
It calculates, the RSD of peak area is 1.42%, that is, shows that chromatographic process repeatability is good, can be used for subsequent experimental.
1 repeated experiment result (n=6) of table
1.3 precision are investigated
(1) withinday precision measures: precision measures method Buddhist nun's aldehyde stock solution of certain volume in 5mL volumetric flask, fixed with acetonitrile
Hold, is configured to each three parts of Buddhist nun's aldehyde solution of various concentration method that basic, normal, high concentration is respectively 10,150,400 μ g/mL.4 DEG C of storages,
In different time points according to step (2) chromatographic condition sample detection, sample introduction is measured five times within one day, records chromatographic peak peak face
Product.
(2) day to day precision measures: precision measures method Buddhist nun's aldehyde stock solution of certain volume in 5mL volumetric flask, fixed with acetonitrile
Hold, is configured to each three parts of Buddhist nun's aldehyde solution of various concentration method that basic, normal, high concentration is respectively 10,150,400 μ g/mL.4 DEG C of storages,
Sample peak area is measured according to step (2) chromatographic condition sample introduction carry out day to day precision investigate, it is each in continuous five days
It same time point sample introduction measurement, records chromatographic peak peak area.
In withinday precision experiment, by the time point in scheme by the method Buddhist nun of respective concentration under step (2) chromatographic condition
Aldehyde solution sample detection, it is as shown in table 2 to measure sample peak area.Go out method Buddhist nun's aldehyde solution low concentration in a few days according to calculated by peak area
Standard deviation is 0.20%, and the in a few days standard deviation of middle concentration is 0.43%, and the in a few days standard deviation of high concentration is 1.12%, table
Bright this method withinday precision is good, meets analysis and requires.
Similarly, in day to day precision experiment, by the time point in scheme by respective concentration under step (2) chromatographic condition
Method Buddhist nun's aldehyde solution sample introduction measures peak area, and the results are shown in Table 3, according to the mark in the daytime of calculated by peak area method Buddhist nun's aldehyde solution low concentration
Quasi- deviation is 0.98%, and the standard deviation in the daytime of middle concentration is 0.82%, and the standard deviation in the daytime of high concentration is 1.06%, is shown
This method day to day precision is good, meets analysis and requires.
2 withinday precision of table investigates result table (n=5)
3 day to day precision of table investigates result table (n=5)
The 1.4 blank rate of recovery
Precision measures certain volume method Buddhist nun's aldehyde stock solution in 5mL volumetric flask, and the blank glue that 0.5mL presses prescription preparation is added
Beam solution after ultrasonic emulsion breaking 5min, with 0.22 μm of filtering with microporous membrane, is configured to low (10 μ g/ with methanol constant volume to graduation mark
ML), method Buddhist nun's aldehyde solution of (150 μ g/mL) in, high (400 μ g/mL) three concentration, each 3 parts of each concentration, by step (2) content
Measuring method measures the content of method Buddhist nun's aldehyde in filtrate, chromatographic peak peak area is recorded, according to gained standard curve (A=15146C-
29860, R2=0.9992) its content and the rate of recovery are calculated.Experimental result is as shown in table 4, method Buddhist nun's aldehyde of high, normal, basic three concentration
Average recovery rate is respectively less than 2% in 98.0%~102%, RSD.The result shows that blank micella is to method Buddhist nun's aldehyde, there is no absorption to make
With not influencing the assay of method Buddhist nun's aldehyde, this method meets measurement and requires.
4 rate of recovery of table investigates result (n=3)
In conclusion the present invention has been successfully established the efficient liquid-phase chromatography method of the external assay of method Buddhist nun's aldehyde, and to this
Method is verified.This method has easy to operate, and specificity is strong, and accuracy is high, precision, the features such as repeatability is good, energy
It is enough effectively to reach the subsequent requirement that assay is carried out to method Buddhist nun's aldehyde, so as to accurately carry out the polymer of method Buddhist nun aldehyde load
The experiment such as encapsulation rate, drugloading rate, release in vitro of micella.
The present invention is not limited to the above-described embodiments, for those skilled in the art, is not departing from
Under the premise of the principle of the invention, several improvements and modifications can also be made, these improvements and modifications are also considered as protection of the invention
Within the scope of.It should be understood that the scope of the present invention be not limited to defined by process, property or component, because of these embodiment party
Case and other descriptions are used for the purpose of schematically illustrating certain aspects of the present disclosure.In fact, this field or related fields
Technical staff obviously the various changes that embodiment of the present invention is made can all be covered within the scope of the appended claims.
Claims (7)
1. a kind of method of method Buddhist nun's aldehyde in high performance liquid chromatography measurement carrier micelle, it is characterised in that: use Agilent-
1260 high performance liquid chromatographs, using UV detector, Detection wavelength 216nm includes the following steps:
(1) chromatographic condition: it is the reverse chromatograms column of filler that chromatographic column, which selects octadecylsilane chemically bonded silica,；Mobile phase is second
The mixture or methanol of nitrile and water and any one of the mixture or acetonitrile of water and 0.01% mixture of phosphoric acid solution；
Wherein: the volume ratio of acetonitrile and water is 60~90:10~40, and the volume ratio of methanol and water is 60~90:10~40, acetonitrile with
The volume ratio of 0.01% phosphoric acid solution is 60~90:10~40；Flow velocity is 0.8~1.2mL/min, and sample volume is 5~15 μ L,
Column temperature is 20~40 DEG C；
(2) Specification Curve of Increasing: precision weighing method Buddhist nun aldehyde (reference substance) in right amount, is placed in volumetric flask, and dilute with flowing phased soln
It releases to scale, as method Buddhist nun's aldehyde standard solution stock solution, then the standard solution stock solution is diluted to method Buddhist nun's aldehyde step by step and contained
Amount is the solution of 10,25,50,100,150,200,250,300,350,400 μ g/mL, presses step (1) described chromatographic condition respectively
Measurement, calculates separately the peak area under same retention time, using content x as abscissa, using peak area y as ordinate, does bid
Directrix curve equation y=f (x), content are 10~400 μ g/mL；
(3) sample to be tested is prepared: precision weighs that sample to be tested is appropriate, add mobile phase dissolved dilution at the solution of debita spissitudo after,
Filtering；
(4) detect the content of method Buddhist nun's aldehyde in method Buddhist nun aldehyde sample to be measured: it is described that method Buddhist nun aldehyde sample solution to be measured is pressed step (1) respectively
Chromatographic condition test, calculates the peak area under same retention time, brings step (2) resulting calibration curve equation into, calculate
The concentration of method Buddhist nun aldehyde sample solution to be measured out, then calculate by the sampling amount of sample the content of method Buddhist nun aldehyde in sample.
2. the method for method Buddhist nun's aldehyde, feature exist in high performance liquid chromatography measurement carrier micelle according to claim 1
In: chromatographic column described in step (1) selects Agilent C18 column 4.6*150mm, 5 μm, Waters C18 column 4.6*150mm, 5 μ
M or Shimadzu C18 column 4.6*150mm, one of 5 μm.
3. the method for method Buddhist nun's aldehyde, feature exist in high performance liquid chromatography measurement carrier micelle according to claim 1
It is preferably the mixture of acetonitrile and water in: step (1) described mobile phase, the volume ratio of the acetonitrile and water is 80:20.
4. the method for method Buddhist nun's aldehyde, feature exist in high performance liquid chromatography measurement carrier micelle according to claim 1
In: step (1) described flow velocity is preferably 1.0mL/min.
5. the method for method Buddhist nun's aldehyde, feature exist in high performance liquid chromatography measurement carrier micelle according to claim 1
In: step (1) described column temperature is preferably 25 DEG C.
6. the method for method Buddhist nun's aldehyde, feature exist in high performance liquid chromatography measurement carrier micelle according to claim 1
In: step (1) sample volume is 10 μ L.
7. the method for method Buddhist nun's aldehyde, feature exist in high performance liquid chromatography measurement carrier micelle according to claim 1
In: step (1) filtering preferably uses 0.22 μm of miillpore filter.
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| CN109966508A (en) * | 2019-04-10 | 2019-07-05 | 西南医科大学 | pH-sensitive targeted polymeric micelles PPi-Far-PMs and their preparation methods and applications |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109966508A (en) * | 2019-04-10 | 2019-07-05 | 西南医科大学 | pH-sensitive targeted polymeric micelles PPi-Far-PMs and their preparation methods and applications |
| CN109966508B (en) * | 2019-04-10 | 2021-11-19 | 西南医科大学 | PH-sensitive targeted polymer micelle PPi-Far-PMs and preparation method and application thereof |
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