CN108918701A - The preparation of hesperetin standard substance and valued methods - Google Patents
The preparation of hesperetin standard substance and valued methods Download PDFInfo
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Abstract
The invention discloses a kind of valued methods of hesperetin standard substance, steps：1) mass percentage that hesperetin in the hesperetin raw material powder is measured using high performance liquid chromatography area normalization method, obtains the purity definite value P of hesperetin raw material powder0；2) moisture, organic volatile impurities and inorganic non-volatile impurity mass percentage in hesperetin raw material powder are measured, in conjunction with the P measured in step 1)0, hesperetin purity definite value P is calculatedHPLC‑AN=P0× [100%-Xw‑Xn‑Xv] × 100%；3) hesperetin purity definite value P is obtained using differential scanning calorimetry methodDSC；4) final hesperetin purity definite value is calculated.Also disclose the preparation method of hesperetin standard substance, step：Standard substance is prepared first：Standard substance candidate is taken, is purified using reverse phase C18 silica gel, is rotated, it is dry, it saves；Purity definite value is carried out according to aforementioned valued methods.
Description
Technical field
The present invention relates to the valued methods of standard substance, and in particular to a kind of preparation of hesperetin standard substance and definite value side
Method.
Background technique
Flavonoids (Flavonoids) is the important a kind of secondary metabolite of plant, it is with reference state (flavonoid glycoside) or certainly
It is present in many food-borne plants such as water fruits and vegetables, beans and tealeaves by state (flavone aglycone) form.Flavonoids is especially more
Methoxy flavone, which has, to be enhanced human immunity, anticancer, inhibiting bacteria and diminishing inflammation, prevents the significantly life such as diabetes and cardiovascular and cerebrovascular disease
Reason effect, therefore, is added in functional health-care foods such as dietary supplements extensively currently as important component.Meanwhile according to
The difference of flavonoid components and content, be also used as characteristic index for citrus geographical sign and the fruit juice product place of production trace to the source with
Authenticity identifies.Although flavonoids is widely used to the fields such as food, medicine and cosmetics, such standard substance is in China
Quantity is few at present, type is single, and the preparation of relevant criterion substance need to be improved with definite value technical level.
Hesperetin, English name Tangeretin, chemical name：5,6,7,8,4'- pentamethoxyl flavones, CAS：481-
53-8.Molecular formula：C20H20O7.Relative molecular mass 372.37Da, colourless crystallization or crystalline powder belong to Polymethoxylated Huang
Ketone, is insoluble in water by 154-156 DEG C of fusing point, is soluble in the organic solvents such as ethyl alcohol, ether.Condition of storage：Cool place is dry.Molecule knot
Structure formula is：
Summary of the invention
The purpose of the present invention is providing in view of the above problems, a kind of properties of samples is stable, characteristic magnitude accurately and reliably tangerine peel
The preparation of plain standard substance and valued methods.
The present invention realize the technical solution that its purpose uses for：
A kind of valued methods of hesperetin standard substance, include the following steps：
1) hesperetin raw material powder is sampled, the hesperetin raw material is measured using high performance liquid chromatography area normalization method
The mass percentage of hesperetin in powder obtains the purity definite value P of hesperetin raw material powder0；
2) biodiversity percentage composition, organic volatile impurities mass percentage and nothing in hesperetin raw material powder are measured
Machine non-volatile impurities mass percentage, in conjunction with the purity definite value P of the hesperetin raw material powder measured in step 1)0,
It is calculated according to following formula I to get hesperetin purity definite value P is arrivedHPLC-AN；
PHPLC-AN=P0× [100%-Xw-Xn-XvThe formula of] × 100% I,
Wherein, XwFor biodiversity percentage composition, XnFor inorganic non-volatile impurity mass percentage, XvIt is waved to be organic
Hair property impurity mass percentage；
3) hesperetin purity definite value P is obtained using differential scanning calorimetry methodDSC；
4) it is calculated according to following formula II to get final hesperetin purity definite value is arrived
4, the valued methods of hesperetin standard substance as described in claim 1, feature exist
In：It further include the analysis of step 5) uncertainty.
In the above-mentioned technical solutions, the chromatographic condition of high performance liquid chromatography area normalization method described in step 1) is as follows：
Chromatographic column：Agilent SB-C18(250mm×4.6mm,5.0μm)；Mobile phase：0.2% acetic acid aqueous solution：The volume ratio of acetonitrile
=45：55；Detection wavelength：375nm；Concentration：1000mg/L；Flow velocity：1.0mL/min；Column temperature：25℃；Sample volume：10μL.
In the above-mentioned technical solutions, it is 148 DEG C that the measuring method of differential scanning calorimetry method, which is initial temperature, in step 3), with
The rate of 0.5 DEG C/min is warming up to 158 DEG C, adds when sample cools down to 30 DEG C of fusing point or less, then with same heating rate
Hot sample obtains the DSC figure of measurement hesperetin purity.
In the above-mentioned technical solutions, hesperetin raw material powder is measured using Karl_Fischer method determination of moisture method in step 2)
Middle biodiversity percentage composition；Organic volatile impurities methanol, ethyl alcohol, acetone, second are measured using headspace GC
Nitrile, ethyl acetate, benzene, toluene, n-hexane mass percentage；It is measured using inductively coupled plasma mass spectrometry inorganic non-
B in volatile impurity, Ti, Cr, Co, Ni, As, Se, Sr, Mo, Cd, Sn, Sb, the content of Ba, using inductively coupled plasma body
Al, Ca, Cu, Fe, K, Mg, Mn, Na, P, the content of Zn in emission spectrographic determination inorganic non-volatile impurity.
A kind of preparation method of hesperetin standard substance, includes the following steps：Standard substance is prepared first：Take hesperetin mark
Quasi- substance candidate is purified using reverse phase C18 silica gel, and eluant, eluent uses the mixture of water and acetone, is rotated after purification, is done
It is dry, it saves；Then purity definite value is carried out to the hesperetin standard substance being prepared according to valued methods above-mentioned.
The purification condition that the preparation method of above-mentioned hesperetin standard substance uses reverse phase C18 silica gel to be purified is reverse phase
Silica gel dosage 150g, applied sample amount 400mg, eluant, eluent ratio is water：Acetone by volume 30:70.
The beneficial effects of the invention are as follows：The preparation method of hesperetin purity rubric substance is provided, hesperetin obtained is pure
Standard substance is spent, long-time stability monitoring in 12 months is completed, properties of samples is stable, characteristic magnitude is accurate and reliable, for related mandarin orange
Tangerine product traceability and authenticity detection, the quality monitoring of health care product, cosmetics and medical product provide technical support.The tangerine of offer
Skin element valued methods use two kinds of different principles of high performance liquid chromatography-area normalization method and differential scanning calorimetry method (DSC)
Method purity definite value is carried out to standard substance raw material, obtained result is very accurate and reliable.
Detailed description of the invention
Fig. 1 is that the Chromatographic Comparison of hesperetin and its impurity schemes after purification.
Fig. 2 is the chromatogram of methanol blank, hesperetin and its impurity under optimal conditions.
Fig. 3 is schemed with the DSC of the rate determination hesperetin purity of 0.5 DEG C/min.
Fig. 4 is hesperetin purity rubric substance definite value uncertainty source analysis and rate the process figure.
Specific embodiment
1 standard substance sample preparation of embodiment
One, prepared by standard substance
Standard substance candidate hesperetin is purchased from Nanjing draft source Biotechnology Co., Ltd, using reverse phase C18 silica gel into
Row purifying, purification condition：Reverse phase silica gel dosage 150g, applied sample amount 400mg, eluant, eluent ratio is water：The volume ratio of acetone is 30:
70.It rotates after purification, using HPLC test sample purity, as shown in Figure 1, the purity of hesperetin is improved by 98.29% after purification
To 99.98%, purification effect is significant.Subsequent dry using baking oven and freeze-drying, moisture is reduced to 0.0251%.Water removal is dry
It after dry, be sufficiently mixed and be uniformly placed in clean, dry packing environment, dispense into 4mL cleaning Agilent Brown Glass Brown glass bottles and jars only, twist
Tightly, it covers.Each Packing Unit is 20mg, and totally 122 bottles, low temperature (4 DEG C) is protected from light, and is saved.
Two, principal component qualitative analysis
Hesperetin standard substance obtained is carried out qualitative point of principal component to candidate using mass spectrography and nucleus magnetic hydrogen spectrum method
Analysis, testing result show that sterling raw material main component is hesperetin.
Three, uniformity testing
It is tested using hesperetin Certified Reference Material Homogeneity of the high-efficient phase chromatogram area normalization method to preparation, liquid phase color
Spectral condition is as follows：Chromatographic column：Agilent SB-C18(250mm×4.6mm,5.0μm)；Mobile phase：0.2% acetic acid aqueous solution：
Acetonitrile=45：55；Detection wavelength：375nm；Flow velocity：1.0mL/min；Column temperature：25℃；Sample volume：10μL.The data of measurement are adopted
With one-way analysis of variance method statistical check, by data statistic analysis it is found that hesperetin purity rubric substance packing unit is uniform,
Meet demand of technical standard.
Four, study on the stability
To investigate hesperetin purity rubric substance under the influence of long-term storage condition and external environment change condition, substance
The ability that physicochemical properties and characteristic magnitude remain unchanged, according to《Standard substance definite value principle and Principle of Statistics》Jie
It continues, long-term and short-term study on the stability has been carried out to hesperetin purity rubric substance using line fitting approach.
1) long-time stability are investigated
According to JJG1006-1994《Primary standard substance technical specification》In requirement, standard substance study on the stability according to
Thin principle after first close.This research is using liquid chromatography respectively in the 0th, 1,3,6,9,12 month progress study on the stability.Often
It is secondary to extract 2 packagings, solution is prepared using weight-volumetric method, each packaging is measured in parallel 3 times, and liquid phase chromatogram condition is as follows：Color
Compose column：Agilent SB-C18(250mm×4.6mm,5.0μm)；Mobile phase：0.2% acetic acid aqueous solution：Acetonitrile=45：55；Inspection
Survey wavelength：375nm；Concentration：1000mg/L；Flow velocity：1.0mL/min；Column temperature：25℃；Sample volume：10μL.Stability test knot
Fruit is as shown in table 1, and to monitoring result fitting a straight line, the slope b known to linear equation1With intercept b0.It is steady that we will continue monitoring
The qualitative validity period to extend standard substance.
1 hesperetin purity rubric substance long-time stability monitoring result (%) of table
2) short-term stability is investigated
Sample is placed in 20 DEG C, 40 DEG C and 60 DEG C insulating boxs (simulation traffic condition) to save, respectively in the 1st, 3,5,7,9
It carries out STABILITY MONITORING, and measuring method is identical as long-time stability monitoring, and liquid phase chromatogram condition is as follows：Chromatographic column：Agilent
SB-C18(250mm×4.6mm,5.0μm)；Mobile phase：0.2% acetic acid aqueous solution：Acetonitrile=45：55；Detection wavelength：375nm；
Concentration：1000mg/L；Flow velocity：1.0mL/min；Column temperature：25℃；Sample volume：10μL.Monitoring data are carried out using trend analysis
Investigate, investigate the results show that this standard substance under 60 DEG C and traffic condition below in 9 days can guarantee purity magnitude stablize,
Concrete outcome is as shown in table 2.
2 hesperetin purity rubric substance short-term stability of table investigates result (%)
| Time (day) | Temperature condition (20 DEG C) | Temperature condition (40 DEG C) | Temperature condition (60 DEG C) |
| 1 | 99.85 | 99.85 | 99.85 |
| 3 | 99.83 | 99.82 | 99.82 |
| 5 | 99.86 | 99.84 | 99.86 |
| 7 | 99.84 | 99.85 | 99.82 |
| 9 | 99.84 | 99.82 | 99.82 |
| Average value | 99.84 | 99.84 | 99.83 |
| b1 | -0.0005 | -0.0015 | -0.0003 |
| b0 | 99.847 | 99.844 | 99.849 |
| s2 | 0.000173 | 0.0003067 | 0.0005067 |
| s(b1) | 0.0020820 | 0.0027690 | 0.0035590 |
| t0.95,n-2 | 3.18 | 3.18 | 3.18 |
| Conclusion | |b1|<t0.95,n-2·s(b1), stablize | |b1|<t0.95,n-2·s(b1), stablize | |b1|<t0.95,n-2·s(b1), stablize |
In conclusion hesperetin purity rubric 12 months long-time stability of substance are good, in 20 DEG C, 40 DEG C, 60 DEG C of mould
Characteristic magnitude is stablized under the conditions of quasi- transport temperature, 9 days haulage times.
2 standard substance definite value of embodiment
Referring to " national standard reference material technology specification ", by literature survey early period and preliminary result, this research is adopted
With the method for two kinds of different principles of liquid chromatography area normalization method and differential scanning calorimetry method to hesperetin purity rubric substance
Definite value.Liquid chromatography area normalization method includes HPLC-UV method measurement hesperetin principal component, karl Fischer coulomb method measurement moisture
Content, Headspace Gas Chromatography volatile impurities content and ICP-MS and ICP-OES measurement inorganic element content etc.,
For the traceability and accuracy for guaranteeing measurement result, the instrument and equipment used is calibrated by measurement verification or standard substance.
Valued methods 1：High performance liquid chromatography-area normalization method
One, high-efficient liquid phase chromatogram condition optimization and measurement
(1) ultraviolet full scan
The ultraviolet full scan of hesperetin, the ultraviolet maximum absorption wavelength of hesperetin have been done before high-efficient liquid phase chromatogram condition optimization
For 325nm, therefore the subsequent wavelength for using 325nm as efficient liquid phase condition optimizing.
(2) chromatographic column optimizes
Optimize efficient liquid phase condition using hesperetin before purification first.Suitable chromatographic column is selected, can be reached more
Good analytical effect.According to the document of access, SB-AQ, SB-C18 and Eclipse Plus C18 chromatographic column are compared for tangerine
The chromatography appearance situation of skin element and its impurity, the liquid-phase condition for optimizing chromatographic column are：Mobile phase：0.2% acetic acid aqueous solution：Acetonitrile
=45:55；Wavelength：325nm；Sample concentration：3000ppm；Flow velocity：1mL/min；Sample volume：5μL；Column temperature：25℃.Use SB-
When AQ column, the impurity of 6.775min and 11.517min or so are closer with main peak, and the response of principal component and peak shape are not so good as
SB-C18；When using Eclipse Plus C18, the peak shape of the main peak of 8.395min or so is too wide, the peak shape of impurity peaks and main peak
All compared with the width in SB-C18 chromatographic column；And when using SB-C18 column, impurity separates preferably with main peak, and peak shape is relatively narrow, therefore selects
SB-C18 chromatographic column.
(3) optimization of organic phase
On this basis, using 0.2% acetic acid aqueous solution as water phase, methanol and acetonitrile are compared as organic phase, hesperetin
And its appearance situation of impurity.Liquid-phase condition：Chromatographic column：SB-C18；Water phase：0.2% acetic acid aqueous solution；Wavelength：325nm；Sample
Product concentration：1000ppm；Flow velocity：1mL/min；Sample volume：5μL；Column temperature：25℃.When methanol is as organic phase, principal component and its
The response of impurity is all lower, and appearance is later, and all peak 120min could be complete out；When acetonitrile is as organic phase, separating effect is obvious
Better than methanol, under equal conditions, chromatographic isolation is more complete, and impurity response is significant, and the peak shape of main peak and all impurity is narrow compared with methanol,
Therefore selecting acetonitrile is organic phase.
(4) optimization of mobile phase ratio
Using 0.2% acetic acid aqueous solution as water phase, acetonitrile is organic phase, continues to optimize tangerine under the conditions of different mobile phase ratios
The appearance situation of skin element and its impurity.When water phase and acetonitrile ratio are 30:When 70, the impurity of 7.077min or so fails and main peak
It is completely separable；When water phase and acetonitrile ratio are 45:When 55, all impurity are separated with main peak, and all peaks can be complete out in 20min；
When water phase and acetonitrile ratio are 55:Impurity is kept completely separate with main peak when 45, but whole appearance time is too long, nearly 50min.Therefore
Mobile phase ratio selects 0.2% acetic acid aqueous solution：Acetonitrile=45:55.
(5) optimization of wavelength
In order to investigate the response of impurity at different wavelengths, 240,310,350,365,375 and 380nm six is had chosen not
Same wavelength, as the result is shown under the wavelength of 375nm, the impurity of 6.47min and 15.688min respond most abundant, the peak of impurity
Area is maximum, therefore selects 375nm for definite value wavelength.
(6) optimization of sample concentration
For impurity responsiveness, tetra- kinds of various concentrations of 100mg/L, 500mg/L, 1000mg/L and 3000mg/L have been investigated
Hesperetin methanol solution chromatography appearance situation, and compared with methanol solvate blank.Concentration is 100mg/L and 500mg/L
When, the impurity of 6.47min and 15.688min are hardly visible, 1000mg/L concentration or more, 6.47min and 15.688min's
Impurity is more apparent, and impurity response is more abundant, therefore selects the concentration of 1000mg/L.
In conclusion finally determining hesperetin liquid phase chromatogram condition is as follows：Chromatographic column：Agilent SB-C18(250mm
×4.6mm,5.0μm)；Mobile phase：0.2% (volumn concentration) acetic acid aqueous solution：Volume ratio=45 of acetonitrile：55；Detection
Wavelength：375nm；Concentration：1000mg/L；Flow velocity：1.0mL/min；Column temperature：25℃；Sample volume：10μL.Methanol under optimal conditions
Blank, hesperetin chromatogram is as shown in Fig. 2, hesperetin main peak and impurity content table are as shown in table 3.Separating degree (also known as resolution ratio)
It may determine that the separation situation of separate substance in the chromatography column, indicated with R, equal to the difference and dichromatism of the retention time at adjacent two peak
The ratio between spectral peak peak width mean value.R<When 1, two peaks partly overlap, and when R=1, separating degree is basically separated up to 98%, two peak；R=1.5
When, separating degree is kept completely separate up to 99.7%, two peak.Under the chromatographic condition, the separating degree of principal component is 7 or more, all impurity
Separating degree all 1.5 or more, separating degree is preferable.Therefore the chromatographic condition meets the chromatography measurement of hesperetin purity rubric substance
It is required that.
The pure hesperetin content table of table 3
| Serial number | Retention time | Peak area (microvolt * seconds) | % area |
| 1 | 6.467 | 505 | 0.07 |
| 2 | 8.797 | 682546 | 99.87 |
| 3 | 15.725 | 401 | 0.06 |
Two, Karl_Fischer method determination of moisture
(1) key instrument and reagent
DL32 type micro-water analyzer (Mei Tele company of Switzerland)；XS105 assay balance (Max=120g, d=
0.01mg, Mei Tele company of Switzerland)；50 μ L micro syringes；Coulomb method anolyte Coulomat AK (it is used for ketone,
Fluka company)；Merck Apura-WaterStandard standard water sample, aqueous 0.1%.
(2) determination step
Karl Fischer coulometric titration instrument is opened, instrument automatic Titration moisture to terminal, keeps not having moisture in titration cell.With
Micro syringe draws 40 μ L standard water samples, and with balance precise quality, the pin hole through gasket injects titration cell, input quality
It is titrated and is calibrated.Not uncapped due to calibration process causes air to enter, and does not need background correction.Claimed with pan paper
The sample of 5-7mg is taken, opens plastic cover on titration cell rapidly, sample addition reaction flask is titrated, sample-adding process control exists
5s or so, input weigh quality, end to be titrated, and system can show calculated water content, each sample replication 5 automatically
It is secondary.In order to eliminate influence of the moisture in air to measurement result, simulation sample-adding process, quality and time, but it is not loaded progress
Measurement calculates the moisture blank value in air, obtains final result after deducting blank.
Three, organic volatile impurities measure
Due to having used acetone as a part of eluant, eluent in purification process, it is contemplated that solvent may be introduced in sample
Impurity acetone, thus using headspace GC determine in hesperetin purity rubric substance including acetone
8 kinds of organic volatile impurities.Specific experiment process and result are as follows.
(1) key instrument and reagent
SHIMADZU gas chromatograph (GC-2018), flame ionization ditector (FID) (Shimadzu), HS-10 type head space
Sample injector (Shimadzu), 20mL headspace sampling bottle (Shimadzu), methanol, ethyl alcohol, acetonitrile, ethyl acetate, acetone, benzene, toluene, n-hexane
(china institute of metrology).
(2) experiment condition and parameter
Chromatographic column DB-624 (0.32mm × 1.8 μm, 30m), carrier gas：Hydrogen flowing quantity：40ml/min, air mass flow：
400ml/min, detector temperature：260℃；Temperature programming condition：Initial 45 DEG C of holdings 5min, with 7 DEG C per minute of rate liter
Temperature is warming up to 190 DEG C to 60 DEG C, then with 15 DEG C of rate per minute.
Sample volume：1mL, split ratio：5:1.
Constant temperature furnace temperature：90℃；Sample flow path temperature：115℃；Transmission line temperature：120℃.
(3) experimental procedure
60mg or so methanol, ethyl alcohol, acetonitrile, ethyl acetate, acetone, benzene, toluene, n-hexane is taken to be dissolved in 10mL capacity respectively
Simultaneously its quality of accurate recording, DMSO constant volume are used as mother liquor (concentration substantially 6000ppm) after mixing in bottle.
Mark is bent to be prepared：It respectively takes 1mL mother liquor in 15mL centrifuge tube, 9mLDMSO is added, mixing is configured to 600ppm's or so
Solution respectively takes the methanol of 1mL600ppm or so, ethyl alcohol, acetonitrile, ethyl acetate, acetone, benzene, toluene in 15mL centrifuge tube, adds
DMSO solvent is made into 7 kinds of mixed marks of about 60ppm to 10mL, then is successively diluted to the mixed mark of 30ppm, 6ppm and 0.6ppm, mixes
It is even.N-hexane is made into single mark, is successively diluted to the single of 60ppm, 30ppm, 6ppm and 0.6ppm by 600ppm and marks, mixes.Dan Biao
It takes 2mL in 20mL ml headspace bottle with the mixed each concentration of mark, is sealed immediately with gland device, it is to be determined.
Sample：The sample of 20mg (being accurate to 0.01mg) is weighed in the ml headspace bottle of 20mL, and accurate recording sample quality,
The DMSO dissolution of 2mL is added, is sealed immediately with gland device, it is to be determined.Volatile solvent information is as shown in table 4.The measurement of sample
As a result substitute into that mark is bent to calculate impurity content, and compared with DMSO blank after it was determined that retention time 2.989min,
The position of 2.582min, 4.234min are respectively acetone, ethyl alcohol, n-hexane.It calculates by the way that mark is bent it is found that hesperetin purity rubric
Acetone in substance, ethyl alcohol, n-hexane dissolvent residual mass fraction average value be 0.001423%, 0.000564%,
0.003597%.Volatile organic solvent impurity total mass fraction is 0.005584%.
4 volatile solvent information of table
Four, inorganic non-volatile impurity determination
(1) inductively coupled plasma mass spectrometry
Orange is detected using micro-wave digestion-inductively coupled plasma mass spectrometry (Microwave-assisted ICP-MS)
B in skin element sample, Ti, Cr, Co, Ni, As, Se, Sr, Mo, Cd, Sn, Sb, Ba content, specific experiment process are as follows.
1) laboratory apparatus and reagent
Icp ms (ICP-MS) match Mo Feishier Co., Ltd ICP-MS X series 2, ten thousand
/ mono- assay balance, purity>99.99% high-purity argon gas, CEM microwave dissolver (MARS SYSTEM), ultrapure water, nitric acid are (excellent
Grade is pure), hydrogen peroxide (excellent pure grade), B, Ti, Cr, Co, Ni, As, Se, Sr, Mo, Cd, Sn, Sb, Ba single element standard solution is made into
Mixed standard solution (10mg/L).
2) experimental method
The foundation of standard working curve：The production of standard working curve is by configuring the mixed mark solution of various concentration
(0.1ppm、1ppm、10ppm、50.0ppm、100.0ppm、500ppm)。
Internal standard solution (Re185, Ge72)：With ultrapure water as medium, wiring solution-forming concentration is 5 μ g/L.
Sample pre-treatments：Microwave Digestion.7 uniform dry samples are weighed, each 0.100g is accurate to 0.0001g, in 55mL
In digestion tube, add 3mL nitric acid, soaked overnight is added 1mL hydrogen peroxide, covers molten sample lid, be placed in pressure pan, place into microwave
In molten sampling device, setting microwave system clears up program, starts to clear up sample.After resolution is fully completed, inner canister is taken out, room is cooled to
Temperature is transferred in 25mL volumetric flask, constant volume with water, is mixed spare.Reagent blank is done simultaneously.Instrument condition：Nebulizer flow
1.02L/min；Scanning times 45 times；Secondary air amount 0.80L/min；Cooling gas flow 13L/min.
(2) inductively coupled plasma emission spectrography
It is examined using micro-wave digestion-inductively coupled plasma emission spectrography (Microwave-assisted ICP-OES)
The Al, Ca, Cu, Fe, K, Mg, Mn, Na, P, Zn in hesperetin sample are surveyed, experimentation is as follows.
1) laboratory apparatus and reagent
5100 ICP-OES of inductive coupling plasma emission spectrograph (ICP-OES) Agilent Technologies,
A ten thousandth assay balance, purity>99.99% high-purity argon gas, air compressor, recirculated cooling water, CEM microwave dissolver
(MARS SYSTEM), ultrapure water, nitric acid (excellent pure grade), hydrogen peroxide (excellent pure grade), Al, Ca, Cu, Fe, K, Mg, Mn, Na, P, Zn
Standard solution.
2) experimental method
The foundation of standard working curve：The production of standard working curve is by configuring the mixed mark solution of various concentration
(0.1ppm、1.0ppm、5.0ppm、10.0ppm、20ppm、40ppm)。
Sample pre-treatments：Microwave Digestion.7 uniform dry samples are weighed, each 0.100g is accurate to 0.0001g, in 55mL
In digestion tube, add 3mL nitric acid, soaked overnight is added 1mL hydrogen peroxide, covers molten sample lid, be placed in pressure pan, place into microwave
In molten sampling device, setting microwave system clears up program, starts to clear up sample.After resolution is fully completed, takes out inner canister and be cooled to room
Temperature is transferred in 25mL volumetric flask, constant volume with water, is mixed spare.Reagent blank is done simultaneously.Instrument condition：Radial view mode；
RF power 1200w；Nebulizer flow 0.70L/min；Secondary air amount 1.00L/min；Plasma flow amount 12L/min.It is logical
The content for crossing each element in the hesperetin that both the above method measures is shown in Table 5.
Non-volatile inorganic element impurity measurement result in 5 hesperetin purity rubric substance of table
After obtaining testing result, calculated according to following formula I to get final hesperetin purity definite value P is arrivedHPLC-AN；
PHPLC-AN=P0× [100%-Xw-Xn-XvThe formula of] × 100% I,
Wherein, P0Purity definite value for the hesperetin raw material powder measured using high performance liquid chromatography area normalization method, Xw
For biodiversity percentage composition, XnFor inorganic non-volatile impurity mass percentage, XvFor organic volatile impurities quality percentage
Content.
According to preceding method and step, the hesperetin purity rubric substance measured by high performance liquid chromatography area normalization method
Mass fraction be 99.81%, specific testing result is shown in Table 6.
The high performance liquid chromatography area normalization Par value result (unit of 6 hesperetin of table：Mass fraction, %)
Valued methods 2：Differential scanning calorimetry method (DSC)
According to ASTME928,98.5% substance is higher than for purity, DSC method is a kind of accurate, quick and convenient pure
Spend measuring method.Its theoretical foundation is Vant Hoff equation, and calculation formula is as follows：
In formula：T0The fusion temperature for the pure material that-impurity content is zero；
TsThe fusion temperature of-sample；
R-gas constant；
ΔHfMole heat content of-sample；
The molar fraction of impurity in x-sample；
The sample score that F-has melted.
Vant Hoff equation shows that the reduction of sample melting temperature is directly proportional to the molar fraction of its impurities, for
For specific sample, T0, R, x and Δ HtIt is certain, TsOnly changed linearly with 1/F.Therefore, the molar fraction x of impurity can
To be calculated by the slope of fitting a straight line.
DSC instrument is calibrated using indium standard substance GBW (E) 120182 before measurement.And optimizing influences hesperetin purity
Heating rate, the purity of hesperetin only has 98.59% when using rate (- 250 DEG C of the room temperature) of 10 DEG C/min for the first time, purity compared with
Low, continuous several times use the rate determination parallel sample of 10 DEG C/min, and hesperetin purity is all very low, with liquid chromatography area normalizing
The hesperetin purity of change method measurement differs greatly.Applicant analysis, possible heating rate is too fast, and purity is caused to decline, it is subsequent
Sample is measured using the heating rate of 0.5 DEG C/min close within the temperature range of fusing point, fusing point nearby two peaks occurs, pure
Degree is risen, and through inspection information, the result fusing point of DSC test nearby has much there are two endothermic peak situation, it may be possible to following
Several situations：1, crystallized crystalline region has begun melting because crystallization is not perfect under lower temperature, meanwhile, it was melting
Cheng Zhong can also be crystallized at a temperature of this, (nucleus is not allowed easily generated under higher temperature, but since the movement of temperature macromolecular chain is active,
Can be crystallized using the crystalline region not melted originally as nucleation site, and then form more orderly crystalline region) and tied again
Brilliant situation；When temperature continues to increase, the crystalline region recrystallized at this time just starts to melt, and then two peaks occurs.2, unstable crystalline substance
The transformation of type.In the crystallization of some polymer, because crystallization temperature is different, different crystal forms, α, β, γ etc. will form, furthermore
It also has a kind of similar with alpha-crystal form, but without its stable crystal form α ' crystalline substance, crystallizes form this crystal form at a lower temperature
Afterwards, α crystalline substance can be transformed into melting process, this is a kind of solid-solid transition process, first melting brilliant by the brilliant direct transformation α of α '
Peak corresponds to the brilliant melting of a small amount of α ', and second endothermic peak correspond to the melting of α brilliant (being come by α ' transformation), goes back among such case is general
Have a small exothermic peak.
Therefore nearby there are two peaks in hesperetin fusing point, may belong to second situation, the transformation of a variety of crystal forms.To understand
Certainly such case keeps definite value more accurate, and the sample for doing experiment is done primary experiment again with identical rate.As a result as schemed
Shown in 3, nearby only there is a peak in fusing point, and hesperetin purity is increased to 99.85%.Therefore the measuring method of subsequent sample all changes
For：Initial temperature is 148 DEG C, is warming up to 158 DEG C with the rate of 0.5 DEG C/min, when sample cools down to 30 DEG C of fusing point or less,
Sample is heated with same heating rate again, obtains the DSC figure of measurement hesperetin purity.Heating sample to fusing point or more for the first time is
In order to allow the crystal form near hesperetin fusing point sufficiently to change, second of measurement purity.
Influence of the heating rate to purity, brilliant when optimizing second of measurement hesperetin purity after crystal form sufficiently changes simultaneously
The heating rate that type sufficiently changes is 1 DEG C/min, investigated respectively measurement purity heating rate be 0.5 DEG C/min, 1 DEG C/
To the influence of hesperetin purity when min and 3 DEG C/min, it is measured in parallel three times.The result shows that with the increase of heating rate, tangerine peel
The purity of element constantly reduces, therefore selects 0.5 DEG C/min for the heating rate of purity definite value.7 samples are measured in parallel, as a result such as
Shown in table 7.
DSC measurement result (the unit of 7 hesperetin purity of table：%)
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 | It is average | SD |
| Purity | 99.82 | 99.83 | 99.79 | 99.85 | 99.79 | 99.82 | 99.85 | 99.82 | 0.02 |
Three, purity definite value result
The result of purity definite value takes the average value of two kinds of different principle method measurement results, as a result as follows：
Wherein,It is final hesperetin purity Coriolis mass score definite value as a result, PHPLC-ANTo use high performance liquid chromatography face
The hesperetin purity Coriolis mass score definite value that product normalization method measures, PDSCThe hesperetin measured for use differential scanning calorimetry method is pure
Spend mass fraction definite value.
In conclusion final hesperetin purity Coriolis mass score definite value result is 99.82%.
3 uncertainty evaluation of embodiment
Hesperetin purity rubric substance uncertainty source analysis and rate the process are as shown in Figure 4.Hesperetin purity rubric
The uncertainty of substance consists of three parts：The uncertainty u that the uniformity of standard substance introducesbb；The stability of standard substance
Uncertainty (the short-term stability u of introducingltsWith long-time stability usts) and standard substance definite value process introduce it is uncertain
Spend uchar。
One, the uncertainty that uniformity introduces
According to demand of technical standard, uniformity assessment is carried out using one-way analysis of variance method.Uniformity standard deviation sH
It can be calculated with formula：
In formula：SH is uniformity standard deviation；For between-group variance；For intra-class variance；N is pendulous frequency in group.
In this case, sHIt is equal to partial uncertainty u caused by inhomogeneities between bottlebb, as formula
ubb=SH=0.00342%.
Two, the uncertainty that stability introduces
According to the data of table 1, the uncertainty u for the long-time stability that validity period is 12 monthsslsIt calculates as follows：
usls=s (b1) X=0.001054 × 12=0.01265%
The uncertainty u introduced according to the data of table 2, short-term stabilitystsIt calculates as follows：
usts=s (b1)·X
At 20 DEG C：
usts1=s (b1) X=0.01874%
At 40 DEG C：
usts2=s (b1) X=0.02492%
At 60 DEG C：
usts3=s (b1) X=0.03203%
So：
Three, the uncertainty that definite value introduces
The uncertainty that liquid chromatography area normalization method definite value introduces
According to liquid chromatography area normalization method definite value process and its measurement parameter it is found that liquid chromatography area normalization method
Uncertainty u (PHPLC-AN) calculation formula is as follows：
Wherein, u (PHPLC-AN) be area normalization method uncertainty, u (P0) be liquid chromatography uncertainty, u
(Xw) be moisture measurement uncertainty, u (Xn) it is the uncertainty that non-volatile impurities measure, u (Xv) it is that volatile impurity is surveyed
The uncertainty of amount.
(1) uncertainty of measurement of liquid chromatogram
1) the uncertainty u1 that liquid chromatogram Par value measurement reproducibility introduces is by the standard deviation calculation that measures,
u1=0.0090%
2) each ingredient uncertainty that response difference introduces under different Detection wavelengths:
In formula, BimaxλFor the largest percentage content of the impurity component i under 6 Detection wavelengths；
BI definite value λFor the degree of impurity component i under definite value wavelength；
u2-iFor the partial uncertainty of impurity component i.
Liquid chromatogram measuring Composite Seismogram is calculated：
(2) uncertainty that moisture introduces
The partial uncertainty very little that balance claims sample to introduce, so the main uncertain source of moisture measurement is the weight of measurement
Renaturation uA, the measurement result X of moisturewIt is 0.0251%, standard deviation 0.0072%, therefore determination of moisture introducing is uncertain
Degree:(Xw)=0.0072%
(3) uncertainty that other impurities introduce
The partial uncertainty very little that balance claims sample to introduce, so the measurement of non-volatile inorganic element impurity is main not true
Determine the repeated u that source is measurementA, the measurement result X of non-volatile inorganic element impuritynIt is 0.00695%, due to part member
The measurement of element is affected by environment, instrument, therefore, is remembered the uncertainty of non-volatile inorganic element impurity in this research
For u (Xn)=0.00695%.
(4) standard uncertainty of area normalization method：
Four, the uncertainty that differential scanning calorimetry introduces
The uncertainty that dsc measurement method introduces includes A class uncertainty and B class uncertainty.A class uncertainty mainly by
The standard deviation of measurement result calculates, i.e. uA=0.02%.
B class uncertainty is measured the variation of each amount by experiment mainly to estimate.By VantHoff equation：
It can obtain：
In formula：Q-sample heat content；
M-sample quality.
So the B class uncertainty that each amount variation of DSC introduces can be estimated by following formula：
In the measurement of DSC method, GBW13202 indium heat analysis standard substance has been used.The given fusing break of certificate is 3265J/
Mol, the actually measured fusing break of instrument are 3340J/mol；So Δ Q/Q=0.023, the fusing point that certificate gives indium are
156.52, uncertainty is 0.01 DEG C, T0-TsIt is 1.44 DEG C, so Δ T/ (T0-Ts)=0.0069；Sample is claimed to use maximum range
150mg/0.001mg assay balance, sample sample weighting amount are 3mg, and the mobility of balance weighing and Uncertainty etc. are estimated as 5
μ g, so, Δ W/W=0.0017；The relative error of peak area is Δ F/F=0.02.Therefore, uB, rel=0.03；uB=0.03
× (1-99.82%)=0.0054%；
To sum up, the uncertainty of DSC introducing is：
Due to two kinds of definite value result equally accurates, the uncertainty calculation that purity definite value introduces is as follows：
Five, the synthesis of standard substance and expanded uncertainty
The Composite Seismogram calculation formula of standard substance is as follows：
Expanded uncertainty is：UCRM=k × uCRM=0.104% (k=2).
Six, results expression
Shown in sum up, the characteristic magnitude and its uncertainty of hesperetin purity rubric substance are as shown in table 8.
8 hesperetin purity rubric substance characteristics magnitude of table and its uncertainty
| Title | Mass fraction/% | Uncertainty/% (k=2) |
| Hesperetin purity rubric substance | 99.8 | 0.2 |
For the hesperetin purity rubric substance that the present invention develops, long-time stability monitoring in 12 months, sample is completed
Matter is stable, characteristic magnitude is accurate and reliable.
Claims (7)
1. a kind of valued methods of hesperetin standard substance, it is characterised in that：Include the following steps：
1) hesperetin raw material powder is sampled, the hesperetin raw material powder is measured using high performance liquid chromatography area normalization method
The mass percentage of middle hesperetin obtains the purity definite value P of hesperetin raw material powder0；
2) biodiversity percentage composition, organic volatile impurities mass percentage and inorganic non-in hesperetin raw material powder are measured
Volatile impurity mass percentage, in conjunction with the purity definite value P of the hesperetin raw material powder measured in step 1)0, according to
Following formula I calculates to arrive hesperetin purity definite value PHPLC-AN；
PHPLC-AN=P0× [100%-Xw-Xn-XvThe formula of] × 100% I,
Wherein, XwFor biodiversity percentage composition, XnFor inorganic non-volatile impurity mass percentage, XvFor volatile organic
Impurity mass percentage；
3) hesperetin purity definite value P is obtained using differential scanning calorimetry methodDSC；
4) it is calculated according to following formula II to get final hesperetin purity definite value is arrived
2. the valued methods of hesperetin standard substance as described in claim 1, it is characterised in that：It further include that step 5) is uncertain
The analysis of degree.
3. the valued methods of hesperetin standard substance as described in claim 1, it is characterised in that：Efficient liquid described in step 1)
The chromatographic condition of phase chromatography area normalization method is as follows：Chromatographic column：Agilent SB-C18(250mm×4.6mm,5.0μm)；Stream
Dynamic phase：0.2% acetic acid aqueous solution：Volume ratio=45 of acetonitrile：55；Detection wavelength：375nm；Concentration：1000mg/L；Flow velocity：
1.0mL/min；Column temperature：25℃；Sample volume：10μL.
4. the valued methods of hesperetin standard substance as described in claim 1, it is characterised in that：Show that residual quantity heat is swept in step 3)
It is 148 DEG C that the measuring method for retouching method, which is initial temperature, is warming up to 158 DEG C with the rate of 0.5 DEG C/min, cools down to sample molten
Or less 30 DEG C when, then with same heating rate heat sample, obtain measurement hesperetin purity DSC figure.
5. the valued methods of hesperetin standard substance as described in claim 1, it is characterised in that：Taken in step 2) using karr
Not biodiversity percentage composition in method determination of moisture method measurement hesperetin raw material powder；It is surveyed using headspace GC
Determine the mass percentage of organic volatile impurities methanol, ethyl alcohol, acetone, acetonitrile, ethyl acetate, benzene, toluene, n-hexane；It adopts
B, Ti, Cr, Co, Ni, As, Se, Sr, Mo, Cd in inorganic non-volatile impurity are measured with inductively coupled plasma mass spectrometry,
The content of Sn, Sb, Ba measure Al, Ca, Cu in inorganic non-volatile impurity using inductively coupled plasma emission spectrography,
The content of Fe, K, Mg, Mn, Na, P, Zn.
6. a kind of preparation method of hesperetin standard substance, which is characterized in that include the following steps：Standard substance is prepared first：
Hesperetin standard substance candidate is taken, is purified using reverse phase C18 silica gel, eluant, eluent uses the mixture of water and acetone, pure
It is rotated after change, it is dry, it saves；
Then the hesperetin standard substance being prepared is carried out according to valued methods described in any one of claim 1 to 5 pure
Spend definite value.
7. the preparation method of hesperetin standard substance as claimed in claim 6, which is characterized in that using reverse phase C18 silica gel into
The purification condition of row purifying is reverse phase silica gel dosage 150g, and applied sample amount 400mg, eluant, eluent ratio is water：Acetone by volume 30:
70。
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| CN113030297A (en) * | 2021-02-19 | 2021-06-25 | 广东省计量科学研究院（华南国家计量测试中心） | 2,2',4,4' -tetrabromodiphenyl ether solution standard substance in methanol and preparation method thereof |
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