CN108918737A - A kind of pre-treating method and efficient liquid-phase chromatography method measuring alkannin in w/o type Asian puccoon skin care milk - Google Patents
A kind of pre-treating method and efficient liquid-phase chromatography method measuring alkannin in w/o type Asian puccoon skin care milk Download PDFInfo
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Abstract
The invention belongs to the technical fields of analytical chemistry, disclose a kind of pre-treating method and efficient liquid-phase chromatography method for measuring alkannin in w/o type Asian puccoon skin care milk.Pre-treating method：W/o type Asian puccoon skin care milk is mixed with aqueous alkali first, demulsification extraction takes buck layer；Then acid is added into buck layer, adjusts pH value of solution to 4~6, organic solvent is added, stands extraction, takes lower organic layer, removes the organic solvent in organic layer, obtains solid alkannin.The present invention is detected solid alkannin wiring solution-forming by high performance liquid chromatography, obtains the content of alkannin in Asian puccoon skin care milk.Pre-treating method of the invention is simple, quick, and emulsion breaking is complete, and the purity is high of alkannin improves liquid chromatographic detection accuracy and detection sensitivity.For method of the invention simultaneously closer to true value, detection effect is more acurrate；The rate of recovery is high, reproducible.
Description
Technical field
The invention belongs to the technical field of analytical chemistry, it is related to the detection of active constituent in daily chemical products, in particular to W/O
The pre-treating method and utilize high effective liquid chromatography for measuring alkannin content that alkannin detects in the Asian puccoon facial treatment milk of type
Method.
Background technique
Asian puccoon be Boraginaceae Lithospermum herbaceos perennial, with cooling and activating blood, anti-inflammatory analgetic, detoxify promoting eruption function,
It is chiefly used in the treatment of the diseases such as blood-heat and toxin exuberance, atropurpureus macula, measles without adequate eruption, sore, eczema, scald.Asian puccoon it is main medicinal
Ingredient is naphthalene Quinone Pigments, mainly has more than 10 kinds of alkannin derivants such as alkannin and Acetylshikonin to constitute, have callus,
Anti-inflammatory, antibacterial, promotion cytothesis, removing toxic substances promoting eruption and inhibition growth of tumour cell and other effects.Wherein, alkannin is fat-soluble
Ingredient, it is not soluble in water, is dissolved in ethyl alcohol, organic solvent and vegetable fat, is soluble in buck, but meets acid then Precipitation.It is existing
The extracting method of alkannin is mostly vegetable fat extraction and subcritical fluid extraction method etc..
Currently, being formulated in the market with the Asian puccoon class cosmetics of many different cultivars mostly with Radix Arnebiae extract and other
Active constituent, functional grease are compounded with emulsifier, and homogenizer homogeneous forms lotion or microemulsion.It is past in practical Asian puccoon skin care milk
It toward complicated component, needs to carry out pre-treatment to sample, is divided alkannin from Asian puccoon skin care milk by the methods of extraction, column chromatography
From, then using the methods of high performance liquid chromatography or mass spectrum progress content analysis.But how pre-treatment is carried out to lotion, extract cream
Active constituent is the technological difficulties that alkannin measures in skin care milk in liquid.Application No. is 201410216490.9 Chinese patent texts
It offers and discloses a kind of preparation method of multiple-effect tea oil zicao soft plaster, in terms of formulation composition, which is water-oil phase
System largely uses beeswax as emulsifier, is dispersed in alkannin uniformly and stably in lotion in system.Application number
201611185354.3 Chinese patent application document discloses a kind of alkanet cosmetic composition and preparation method thereof, former
Material formula includes：20~30 parts of Asian puccoon root extract, 35~50 parts of Porcine HGF, 1~5 part of SOD, 1~5 part of active peptide,
8~12 parts of squalene, Cer AP~10 part, 12~20 parts of carboxymethyl cellulose, 20~35 parts of lyase liquid and deionized water
In right amount.Because its formula uses water soluble ingredient (ingredients such as polypeptide, enzyme, polysaccharide) and liposoluble constituent (Asian puccoon root extract, angle
Squalene, ceramide), which uses microcapsules technology, Asian puccoon root extract and other group of object ingredient is wrapped up, behavior
Microemulsified product.The Chinese patent literature of application number 201310150896.7 discloses a kind of lithospermum mask pad pasting lotion, face
Alkanet extracting solution and other group of object components utilising micro-emulsion technology are developed a Asian puccoon face by pad pasting and preparation method thereof
Film patch uses lotion.On the whole, on the market Asian puccoon cosmetics are various in style, but there is no to functional component alkannin mostly
Assay is carried out, is caused irregular in product quality and effect.Therefore, in the real application research of alkannin, how
It is simple from complicated multicomponent sample, easily extract alkannin, and separation detection is carried out to it, this link is to daily chemical industry
Control of product quality it is particularly important.
The present invention is directed to the Asian puccoon skin care milk of W/O water-in-oil system, provides a kind of simple, convenient and fast measurement w/o type Asian puccoon
The pre-treating method of alkannin content and the method using high effective liquid chromatography for measuring alkannin content in skin care milk.The present invention
Alkannin is extracted from complex component using simple, convenient and fast pre-treating method, the alkannin rate of recovery of extraction is high, to detection
As a result interference is small；Then using the content of high performance liquid chromatograph detection alkannin.Method of the invention is simple, detection
Accuracy and high sensitivity.
Summary of the invention
In order to fill up the blank of alkannin detection technique in Asian puccoon skin care milk, while for quick, convenient, accurate measurement
The content of alkannin in Asian puccoon skin care milk, the purpose of the present invention is to provide alkannins in a kind of measurement w/o type Asian puccoon skin care milk
The pre-treating method and efficient liquid-phase chromatography method of content.The present invention effectively removes possibility by carrying out pre-treatment to skin care milk
The factor of interference detection result, improves detection accuracy and detection sensitivity, improves the recovery rate and the rate of recovery of alkannin.
The present invention will extract to obtain the progress high performance liquid chromatography measurement of alkannin sample by pre-treatment, and this method is efficiently, quickly, just
It is prompt.
The purpose of the present invention is achieved by the following technical programs：
A kind of pre-treating method measuring alkannin in w/o type Asian puccoon skin care milk, includes the following steps：
(1) w/o type Asian puccoon skin care milk is mixed with aqueous alkali, demulsification extraction takes buck layer；The aqueous alkali is hydrogen
Sodium hydroxide solution or potassium hydroxide solution；
(2) acid is added into buck layer, adjusts pH value of solution to 4~6, organic solvent is added, stands extraction, takes lower layer organic
Layer removes the organic solvent in organic layer, obtains solid alkannin.
Demulsification extraction described in step (1) carries out under conditions of high speed homogenization or ultrasound；
The high speed homogenization is preferably high speed shear homogenizer.
The revolving speed of the high speed homogenization is 5000~15000rpm, and the time of high speed homogenization is 5~15min；
The condition of the ultrasound is：Ultrasonic power is 450~1500W, and supersonic frequency is 15~35KHz, ultrasonic time 120
~300s；
It is described ultrasound be preferably ultrasonic cell disruptor, amplitude transformer can match end diameter be Φ 6, Φ 2, Φ 3, Φ 8,
Φ 10 etc..
In step (1), after demulsification extraction, product is centrifuged, buck layer is isolated；Made using centrifugation containing alkannin
Buck layer is separated with the Material of skin care milk；Material is located at bottom.
The revolving speed of centrifugation is 1000~6000r/min, and centrifugation time is 10~30min.
The concentration of aqueous alkali described in step (1) is 1wt%~10wt%；
The number of demulsification extraction is 3~5 times in step (1), and the dosage of aqueous alkali is w/o type Asian puccoon shield when extraction every time
5~20 times of skin breast matter amount.
Acid described in step (2) is hydrochloric acid, and sour concentration is 5wt%~10wt%.
It is 4~6 that benefit, which is adjusted with acid lye pH, in step (2), and preferably pH is 5.0.
Organic solvent described in step (2) is carbon tetrachloride, chloroform, methylene chloride, ethyl acetate, butyl acetate or toluene
In more than one.
The number of extraction described in step (2) is 3~5 times, and the dosage of organic solvent is molten after acid is added when extraction every time
0.1~3 times of liquid quality, the time of standing are 10~30min.
Removal organic solvent described in step (3), which refers to, carries out decompression rotary evaporation removal organic solvent for organic layer, subtracts
The condition for pressing rotary evaporation is 20~45 DEG C, negative pressure.
In order to measure alkannin content in skin care milk, above-mentioned pre-treating method is obtained into solid alkannin and uses efficient liquid phase
Chromatography is detected.First by solid alkannin wiring solution-forming, then detected using high performance liquid chromatography.
Based on the sample-pretreating method, the present invention also provides alkannins in a kind of liquid chromatographic detection Asian puccoon skin care milk
Measuring method, it is accurate to draw analyte sample fluid note using column chromatography separation technology using methanol and phosphate aqueous solution as mobile phase
Enter high performance liquid chromatograph and carry out chromatographic isolation, using ultraviolet absorption detector, the absorption of alkannin is detected at 515nm wavelength
Value, using external standard method, according to absorption its content of calculated by peak area.
The efficient liquid-phase chromatography method of alkannin, includes the following steps in the measurement w/o type Asian puccoon skin care milk：
(a) by m0The w/o type Asian puccoon skin care milk of quality is handled according to pre-treating method, obtains solid alkannin；It adopts
With methanol by solid alkannin wiring solution-forming, sample to be tested is obtained, the volume of sample to be tested is V；
(b) alkannin standard sample (purity >=98%) is utilized, configures a series of standard solution of various concentrations, passes through height
The measurement of effect liquid phase chromatogram method, establishes standard curve, and make equation of linear regression；
(c) alkannin in sample to be tested is detected using high performance liquid chromatograph, obtains chromatogram；
(d) alkannin standard curve and equation of linear regression are utilized, using external standard method, according to sample to be tested alkannin peak face
Product calculates concentration C of the alkannin content, that is, alkannin in sample to be tested in sample to be tested；
(e) content according to alkannin in sample to be tested and the volume of sample to be tested calculate m0The w/o type of quality is purple
Alkannin content in careless skin care milk.
In step (e) in w/o type Asian puccoon skin care milk alkannin content calculation formula：
C：The concentration of alkannin, mg/mL in sample to be tested；
V：The volume of sample to be tested, mL；
m0：The sampling amount of Asian puccoon cold cream comprising alkannin, mg.
Sample to be tested specifically refers in step (a)：Using methanol dissolved solid alkannin and constant volume, membrane filtration is obtained
Pretreatment liquid；It recycles methanol to be diluted, is diluted to 1~100 times of pretreatment liquid, obtain sample to be tested；
Standard curve described in step (b) is established：(b1) alkannin standard items are weighed, methanol (chromatographically pure) dissolves and determines
Hold, obtains the alkannin standard solution of 1mg/mL；(b2) serial dilutions are used, the alkannin standard solution of 1mg/mL is diluted
At the alkannin standard solution of 0.01,0.02,0.04,0.06,0.08,0.10mg/mL, examined using high performance liquid chromatography sample introduction
It surveys, using sample introduction concentration as abscissa, peak area is that ordinate establishes standard curve, makes the linear of peak area and alkannin concentration
Regression equation.The equation of linear regression of peak area and alkannin concentration：S=1564880C-18300, coefficient R=0.987.
The condition of detection described in step (c) is：
Chromatographic column：SHIMADZU InertSustain C18 column, 5 μm, 250 × 4.6mm；
Mobile phase：Methanol -0.1% phosphate aqueous solution (volume ratio 75:25)；
Flow velocity：0.6~1.0mL/min；
Column temperature：25~35 DEG C；
Detection wavelength：515nm；
Sample volume：1~10 μ L.
The present invention provides the sample-pretreating method of alkannin in the Asian puccoon skin care milk for measuring different formulations system,
The problem for overcoming the demulsification difficulty in extraction process eliminates in Asian puccoon facial treatment milk other complicated ingredients to alkannin
Interference, extracts alkannin from complicated Asian puccoon lotion.And the process of the pre-treatment is simple, quick, helps to realize daily use chemicals
Control of product quality of the enterprise to Asian puccoon skin care item.
Sufficient buck is added into the formula system Asian puccoon skin care milk of W/O Water-In-Oil, W/O water-in-oil emulsifier cannot be by water
Soluble components package, while utilizing the demulsification of high shear dispersion machine or ultrasonic equipment (such as ultrasonic cell disruptor, ultrasonic wave
Cell crushing instrument, ultrasonic wave nano material are crushed instrument, ultrasonic extraction instrument, ultrasonic extractor etc.) demulsification processing is carried out, make
Oil-soluble ingredients and water soluble ingredient quick separating.Water soluble ingredient in substance system is dissolved in buck, though and alkannin is liposoluble
Property ingredient, but it is also soluble in buck.Therefore, alkannin ingredient can separating and extracting goes out from lotion by buck, is dissolved in buck
(blue-violet solution is presented) in layer, separates alkannin with organic liposoluble constituent.It, can be by the alkali containing alkannin using centrifugal treating
Water is separated with lotion Material.Lotion Material is deposited in centrifugation bottom of the tube after centrifugation, takes upper layer buck layer.The present invention is molten using hydrochloric acid
Liquid adjusts pH value of solution to 4~6, recycles organic solvent extraction, can alkannin be separated with water soluble ingredient.This hair
For bright pre-treating method closer to true value, detection effect is more acurrate.
Compared with prior art, the present invention has the following advantages that：
(1) pre-treating method of the invention utilizes addition for the Asian puccoon facial treatment milk of existing W/O water-in-oil system
Buck and high shear dispersion machine or ultrasonic equipment realize the fast emulsion breakingization processing of lotion, complete to Asian puccoon in samples of latex
The extraction of element, alkannin can be released utmostly from emulsion particle.
(2) present invention is separated the ingredient containing alkannin using aqueous alkali, adjusts solution using hydrochloric acid solution
PH to 4.0~6.0 recycles the organic solvents such as methylene chloride to extract from aqueous solution so that alkannin forms tiny precipitating
Alkannin makes test result close to true value.Therefore, for pre-treating method of the invention closer to true value, detection effect is more quasi-
Really.Pre-treating method of the invention further improves inspection from the factor removal that will likely interfere detection result to the greatest extent
Survey accuracy and detection sensitivity.
(3) rate of recovery of the detection method of alkannin reaches 95.0%~104.8% in Asian puccoon facial treatment milk of the invention,
Relative standard deviation (RSDs) is respectively less than 5%, has the characteristics that reproducible, the rate of recovery is high, is highly suitable for various formulas
The detection of alkannin in the Asian puccoon lotion of system.
(4) breaking method of the invention is applied outer using the aqueous alkali more than emulsifier load capacity in W/O system
Power makes w/o type lotion complete demulsification using the high shear forces of high shear dispersion machine, strong cavitation and collision effect
Processing；Or apply outer field energy, using the ultrasonic field action of ultrasonic equipment, cavitation is generated in a liquid, is formed high-strength
It spends shearing force and high-frequency alternating hydraulic pressure is strong, w/o type lotion is made to complete demulsification processing.
Detailed description of the invention
Fig. 1 is the chromatogram of alkannin standard sample (0.04mg/mL alkannin standard specimen)；
Fig. 2 is the standard curve for the alkannin sample that concentration is 0.01~0.1mg/mL；
Fig. 3 is the chromatogram of Asian puccoon skin care milk in embodiment 1；
Fig. 4 is the chromatography that Asian puccoon skin care milk sample passes through pre-treatment and tested by high performance liquid chromatography in embodiment 1
Figure.
Specific embodiment
The method of the present invention is further illustrated in the following with reference to the drawings and specific embodiments.Following embodiment and attached drawing are only used for reality
Example property explanation, is not considered as limiting the invention.Unless specifically indicated, reagent principle used in following embodiments is normal
The reagent raw material that commercially available or commercial sources obtain is advised, unless stated otherwise, method and apparatus used in following embodiments is this
The conventional use of method and apparatus in field.For convenience of description, instrument and reagent used in the embodiment of the present invention are described as follows, but
Therefore the present invention is not limited.
Instrument：
(1) high performance liquid chromatograph HPLC (SHIMADZU LC-20AT) is prepared：Deaerate pump, quaternary pump, automatic sampling
Device, column oven and UV detector；
(2) ultrapure water system (Millipore Reference)；(3) assay balance (Sai Duolisi BSA-224S)；(4)
Desk-top pH meter (plum Teller-support benefit FE20K)；
(5) rotary vacuum evaporator, configuration：Vacuum pump, water-bath, low-temperature circulating system (German IKA-RV10
Digital)；
(6) high shear dispersion machine (German IKA-T18)；(7) digital display mechanical stirring (German IKA-RW20)；(8) vacuum is taken out
Filter pump (gives magnificent III type of instrument SHZ-D in Gongyi City)；(9) ultrasonic cleaner (sound man of virtue and ability SCQ-6201C)；(10) supersonic cell is broken
Broken machine (the new sesame JY92-IIN in Ningbo)；(11) desk-top high-speed refrigerated centrifuge (Sigma 3K15)；(12) various laboratories are general
Glassware.
Reagent：
Alkannin analytical standard product, purity >=98% (aladdin)；HPLC chromatogram is pure：Methanol；
Analytical reagents：Chloroform, methylene chloride, carbon tetrachloride, toluene, ethyl acetate, phosphoric acid, hydrochloric acid, sodium hydroxide；
0.45 μm of organic filter membrane and water phase filter membrane.
Now for the sample pre-treatments of alkannin detection in baby's camellia oil Asian puccoon skin care milk of a W/O water-in-oil system
Method gives 3 embodiments.The formula of this baby's camellia oil Asian puccoon skin care milk is as shown in table 1.
The formula table of baby's camellia oil Asian puccoon skin care milk of 1 W/O water-in-oil system of table
| INCI title | Percentage/% |
| - 2 isostearate of polyglycereol | 3.50 |
| Isohexadecane | 10.00 |
| Nexbase 2004 dimethyl silicone polymer | 0.50 |
| Camellia (CAMELLIA JAPONICA) seed oil | 5.00 |
| Lithospermum euchromum Royle (ARNEBIA EUCHROMA) extract | 6.00 |
| Tocopherol acetate | 0.10 |
| Butyrospermum parkii (BUTYROSPERMUM PARKII) fruit rouge | 0.50 |
| Vaseline | 4.00 |
| Cocoa (THEOBROMA CACAO) seed rouge | 2.00 |
| Zinc oxide | 6.00 |
| Poly- hydroxy stearic acid | 1.50 |
| Glycerol | 15.00 |
| Natrulon H-10 | 4.00 |
| Sodium chloride | 1.00 |
| Decoyl hydroximic acid | 0.10 |
| Pungent glycol | 0.25 |
| Sensiva SC50 | 0.10 |
| 1,3-PD | 0.55 |
| Water | 39.90 |
Embodiment 1
Alkannin contains in baby's camellia oil Asian puccoon skin care milk of the present embodiment for measuring this W/O water-in-oil system
Amount.
The pre-treating method using alkannin content in high effective liquid chromatography for measuring w/o type Asian puccoon skin care milk：
(1) precise 5g Asian puccoon skin care milk sample, addition 30g concentration are 5wt%NaOH solution, high speed shear (shearing
Rate is 8000rpm) 10min, the completion demulsification extraction of w/o type Asian puccoon skin care milk, 4000r/min revolving speed is centrifuged 15min, and (Material is heavy
Form sediment in aqueous alkali bottom), upper layer buck layer (blue-violet solution) is taken, it is 5wt%NaOH that 30g concentration is added into lower sediment
Solution, high speed shear, centrifugation are so repeated 3 times, and merge the buck layer of 3 demulsification extractions；
(2) buck is placed on separatory funnel, 10wt%HCl solution is added, adjust solution ph to 5.6, solution is in pink colour；
Chloroform is added to extract 4 times, the dosage of each chloroform is 30g, takes lower layer's chloroform layer, merges the chloroform layer of 4 extractions, utilizes decompression
(- 0.1MPa negative pressure, 30 DEG C of temperature) is concentrated in Rotary Evaporators, removes chloroform, obtains alkannin solid；
(3) to alkannin solid be added methanol (chromatographically pure) dissolve and be settled in 25mL volumetric flask, use aperture for
0.45 μm of membrane filtration is to get the pretreatment solution X-1 for detecting alkannin.
Embodiment 2
Alkannin contains in baby's camellia oil Asian puccoon skin care milk of the present embodiment for measuring this W/O water-in-oil system
Amount.
The pre-treating method using alkannin content in high effective liquid chromatography for measuring w/o type Asian puccoon skin care milk：
(1) precise 5g Asian puccoon skin care milk sample, addition 35g concentration are 3wt%NaOH solution, high speed shear (shearing
Rate is 9000rpm) 10min, the completion demulsification extraction of w/o type Asian puccoon skin care milk, 5000r/min revolving speed is centrifuged 13min, and (Material is heavy
Form sediment in aqueous alkali bottom), upper layer buck layer (blue-violet solution) is taken, it is 3wt%NaOH that 35g concentration is added into lower sediment
Solution, high speed shear, centrifugation are so repeated 3 times, and merge the buck layer of 3 demulsification extractions；
(2) buck is placed on separatory funnel, 10wt%HCl solution is added, adjust solution ph to 5.4, solution is in pink colour；
Chloroform is added to extract 5 times, the dosage of each chloroform is 35g, takes lower layer's chloroform layer, merges the chloroform layer of 5 extractions, utilizes decompression
(- 0.1MPa negative pressure, 31 DEG C of temperature) is concentrated in Rotary Evaporators, removes chloroform, obtains alkannin solid；
(3) to alkannin solid be added methanol (chromatographically pure) dissolve and be settled in 25mL volumetric flask, use aperture for
0.45 μm of membrane filtration is to get the pretreatment solution X-2 for detecting alkannin.
Embodiment 3
Alkannin contains in baby's camellia oil Asian puccoon skin care milk of the present embodiment for measuring this W/O water-in-oil system
Amount.
The pre-treating method using alkannin content in high effective liquid chromatography for measuring w/o type Asian puccoon skin care milk：
(1) precise 5g Asian puccoon skin care milk sample, addition 40g concentration are 4wt%NaOH solution, ultrasonic emulsion breaking (ultrasound
Power is 1000W, and supersonic frequency is the ultrasonic cell disrupte machine (amplitude transformer end diameter Φ 6) of 25KHz, ultrasonic time 120s),
W/o type Asian puccoon skin care milk completes demulsification extraction, and 3000r/min revolving speed is centrifuged 20min (Material is deposited in aqueous alkali bottom), takes
Upper layer buck layer (blue-violet solution), it is 5wt%NaOH solution that 30g concentration is added into lower sediment, and high speed shear is centrifuged,
It is so repeated 3 times, merges the buck layer of 3 demulsification extractions；
(2) buck is placed on separatory funnel, 7wt%HCl solution is added, adjust solution ph to 5.2, solution is in pink colour；
Methylene chloride is added to extract 4 times, the dosage of each methylene chloride is 40g, takes lower layer's dichloromethane layer, merges the two of 4 extractions
Chloromethanes layer removes methylene chloride, obtains alkannin using decompression Rotary Evaporators concentration (- 0.1MPa negative pressure, 32 DEG C of temperature)
Solid；
(3) to alkannin solid be added methanol (chromatographically pure) dissolve and be settled in 25mL volumetric flask, use aperture for
0.45 μm of membrane filtration is to get the pretreatment solution X-3 for detecting alkannin.
The high performance liquid chromatography of alkannin content in pretreatment solution (sample to be tested) X-1, X-2, X-3 of Examples 1 to 3
Detection method is as follows：
(1) configuration standard solution establishes standard curve：100mg alkannin standard items (purity >=98%) are weighed, are accurate to
0.1mg is settled to 100mL volumetric flask after methanol dissolution, is made into the alkannin standard solution of 1mg/mL, and sufficiently oscillation shakes up；It will
The alkannin standard solution of 1mg/mL utilize methanol dilution, be configured to concentration be 0.01,0.02,0.04,0.06,0.08,
The alkannin standard solution of 0.10mg/mL is detected using 5 μ L of high performance liquid chromatography sample introduction.The appearance time of alkannin is
9.1min.Using the concentration of alkannin standard specimen as abscissa, peak area is that ordinate establishes standard curve, equation of linear regression S
=1564880C-18300, coefficient R=0.987.
(2) Asian puccoon skin care milk pretreatment solution X-1, X-2, X-3 that above-mentioned pre-treatment obtains are utilized respectively efficient liquid phase
5 μ L of chromatography HPLC (SHIMADZU LC-20AT) sample introduction detection, obtains spectrogram；
(3) external standard method and equation of linear regression S=1564880C-18300 are used, according in sample to be tested (pretreatment liquid)
The peak area S of alkannin calculates alkannin content C；
(4) according to formulaCalculate the content ω of alkannin in Asian puccoon skin care milk actual sample；
C in formula：The concentration of alkannin, mg/mL in sample to be tested；
V：The volume of sample to be tested (i.e. pretreatment liquid), mL；
m0：The sampling amount of Asian puccoon skin care milk, mg in Examples 1 to 3.
The testing conditions of high performance liquid chromatography described in step (1)~(2) are：
Chromatographic column：SHIMADZU InertSustain C18 column, 5 μm, 250 × 4.6mm；
Mobile phase：Methanol -0.1% phosphate aqueous solution (volume ratio 75:25)；
Flow velocity：1.0mL/min；
Column temperature：25℃；
Detection wavelength：515nm；
Sample volume：5μL.
The chromatogram of alkannin standard sample (0.04mg/mL alkannin standard specimen) is as shown in Figure 1；Concentration be 0.01~
The standard curve of the alkannin sample of 0.1mg/mL is as shown in Figure 2；The chromatogram of Asian puccoon skin care milk is as shown in Figure 3 in embodiment 1；
Asian puccoon skin care milk sample is by pre-treatment and as shown in Figure 4 by the chromatogram of high performance liquid chromatography test in embodiment 1.
As a result with analysis
The present invention provides the sample pre-treatments modes that alkannin in the Asian puccoon facial treatment milk of W/O water-in-oil type detects.It is real
The sample pre-treatments mode for applying alkannin in the Asian puccoon facial treatment milk that example 1~3 is w/o type, utilizes addition buck and high shear
Dispersion machine or Ultrasonic cell smash realize the demulsification processing of lotion, recycle hydrochloric acid, organic solvent extraction, complete pair
The extraction of alkannin in samples of latex obtains pretreatment solution X-1, X-2, X-3 for detecting alkannin.Utilize efficient liquid phase
The content of alkannin in Asian puccoon lotion is calculated in chromatograph, alkannin standard curve and equation of linear regression, experimental result is shown in
Table 2.In the Asian puccoon facial treatment milk of w/o type formula alkannin testing result >=77.75ng/g, with true value 79.80ng/g
Relative error be respectively less than 3%.
The testing result of alkannin in the Asian puccoon facial treatment milk of 2 different formulations of table
The verifying of method
1. minimum detectability and minimum quantitative limit
The standard solution of a certain amount of alkannin is added in Asian puccoon samples of latex (such as Asian puccoon skin care in embodiment 1
Lotion), make after addition in Asian puccoon lotion the concentration of alkannin be respectively 100,150,200,250ng/g, implement according to the present invention
The sample-pretreating method of alkannin and detection method carry out real in high performance liquid chromatography detection Asian puccoon facial treatment milk described in example 1
It tests.
It is the minimum detectability of the substance according to signal-to-noise ratio S/N >=3, the detection limit of alkannin is in Asian puccoon facial treatment milk
10ng/g is the minimum detectability of the substance according to signal-to-noise ratio S/N >=10, and the detection limit of alkannin is in Asian puccoon facial treatment milk
30ng/g。
2. accuracy
The Asian puccoon facial treatment milk sample (embodiment 1) (being accurate to 0.1mg) of three parts of 1g of successive precise, and number respectively
S-1,S-2,S-3；Pre-treatment and detection are carried out to sample according to 1 the method for the embodiment of the present invention, to described S-1, S-2, S-3
Asian puccoon facial treatment milk sample is measured in parallel, and analysis the results are shown in Table 3, and the relative standard deviation of alkannin is in Duplicate Samples
1.61%；Further, first sample S-1 is carried out replication 5 times, analysis the results are shown in Table 4, reperformance test it is opposite
Standard deviation is 0.92%.
The parallel testing of 3 sample of table
The reperformance test of 4 sample of table
3. the rate of recovery
The standard solution of alkannin is added in blank Asian puccoon facial treatment milk sample, control spiked levels are respectively 50,
100,200ng/g (i.e. the sample of alkannin known concentration) is detected according to high performance liquid chromatography described in the embodiment of the present invention 1
The sample-pretreating method of alkannin and detection method are tested in zicao soft plaster, and 6 parallel surveys are carried out under each spiked levels
Examination, is averaged, and according to practical scalar quantity and testing result, calculates the recovery of standard addition of zicao soft plaster sample, and it is (corresponding real to be shown in Table 5
Apply example 1).The recovery of standard addition of sample is in 95.0%~104.8% range in embodiment 1.
The parallel determination of 5 mark-on sample of table
| Scalar quantity (ng/g) | 25 | 50 | 100 |
| Practical measurement average value | 24.74±1.21 | 48.37±4.73 | 104.84±2.42 |
| The rate of recovery (%) | 95.0±4.84 | 96.7±9.46 | 104.8±2.42 |
Claims (10)
1. the pre-treating method of alkannin in a kind of measurement w/o type Asian puccoon skin care milk, it is characterised in that：Include the following steps：
(1) w/o type Asian puccoon skin care milk is mixed with aqueous alkali, demulsification extraction takes buck layer；
(2) acid is added into buck layer, organic solvent is added in adjusting pH value of solution to 4~6, and extraction is stood, lower organic layer is taken,
The organic solvent in organic layer is removed, solid alkannin is obtained.
2. measuring the pre-treating method of alkannin in w/o type Asian puccoon skin care milk according to claim 1, it is characterised in that：Step
Suddenly demulsification extraction described in (1) carries out under conditions of high speed homogenization or ultrasound；
Aqueous alkali described in step (1) is sodium hydroxide solution or potassium hydroxide solution；
The concentration of aqueous alkali described in step (1) is 1wt%~10wt%；
Acid described in step (2) is hydrochloric acid, and sour concentration is 5wt%~10wt%.
3. measuring the pre-treating method of alkannin in w/o type Asian puccoon skin care milk according to claim 2, it is characterised in that：
The revolving speed of the high speed homogenization is 5000~15000rpm, and the time of high speed homogenization is 5~15min；
The condition of the ultrasound is：Ultrasonic power be 450~1500W, supersonic frequency be 15~35KHz, ultrasonic time 120~
300s。
4. measuring the pre-treating method of alkannin in w/o type Asian puccoon skin care milk according to claim 1, it is characterised in that：
In step (1), after demulsification extraction, product is centrifuged, buck layer is isolated；
Organic solvent described in step (2) is one in carbon tetrachloride, chloroform, methylene chloride, ethyl acetate, butyl acetate or toluene
Kind or more；
PH described in step (2) is 5.0.
5. measuring the pre-treating method of alkannin in w/o type Asian puccoon skin care milk according to claim 4, it is characterised in that：
The revolving speed of centrifugation is 1000~6000r/min, and centrifugation time is 10~30min.
6. measuring the pre-treating method of alkannin in w/o type Asian puccoon skin care milk according to claim 1, it is characterised in that：Step
Suddenly the number of demulsification extraction is 3~5 times in (1), and the dosage of aqueous alkali is w/o type Asian puccoon skin care milk quality when extraction every time
5~20 times；
The number of extraction described in step (2) is 3~5 times, and the dosage of organic solvent is that solution matter after acid is added when extraction every time
0.1~3 times of amount, the time of standing are 10~30min.
7. the efficient liquid-phase chromatography method of alkannin in a kind of measurement w/o type Asian puccoon skin care milk, it is characterised in that：Including following step
Suddenly：
(a) by m0The w/o type Asian puccoon skin care milk of quality is handled according to pre-treating method, obtains solid alkannin；Using methanol
By solid alkannin wiring solution-forming, sample to be tested is obtained, the volume of sample to be tested is V；The pre-treating method is claim 1
~6 described in any item pre-treating methods；
(b) alkannin standard sample is utilized, a series of standard solution of various concentrations is configured, is surveyed by high performance liquid chromatography
It is fixed, standard curve is established, and make equation of linear regression；
(c) alkannin in sample to be tested is detected using high performance liquid chromatograph, obtains chromatogram；
(d) alkannin standard curve and equation of linear regression are utilized, using external standard method, according to sample to be tested alkannin peak area meter
Calculate concentration C of the alkannin content, that is, alkannin in sample to be tested in sample to be tested；
(e) content according to alkannin in sample to be tested and the volume of sample to be tested calculate m0The w/o type Asian puccoon of quality is protected
Alkannin content in skin cream.
8. measuring the efficient liquid-phase chromatography method of alkannin in w/o type Asian puccoon skin care milk according to claim 7, feature exists
In：In step (e) in w/o type Asian puccoon skin care milk alkannin content calculation formula：
C：The concentration of alkannin, mg/mL in sample to be tested；
V：The volume of sample to be tested, mL；
m0：The sampling amount of Asian puccoon cold cream comprising alkannin, mg.
9. measuring the efficient liquid-phase chromatography method of alkannin in w/o type Asian puccoon skin care milk according to claim 7, feature exists
In：Sample to be tested specifically refers in step (a)：Using methanol dissolved solid alkannin and constant volume, membrane filtration obtains pre-treatment
Liquid；It recycles methanol to be diluted, is diluted to 1~100 times of pretreatment liquid, obtain sample to be tested；
Standard curve described in step (b) is established：(b1) alkannin standard items, methanol dissolution and constant volume are weighed, 1mg/mL is obtained
Alkannin standard solution；(b2) serial dilutions are used, the alkannin standard solution of 1mg/mL is diluted to 0.01,0.02,
0.04, the alkannin standard solution of 0.06,0.08,0.10mg/mL, using high performance liquid chromatography sample detection, with sample introduction concentration
For abscissa, peak area is that ordinate establishes standard curve, makes the equation of linear regression of peak area and alkannin concentration.
10. measuring the efficient liquid-phase chromatography method of alkannin in w/o type Asian puccoon skin care milk, feature according to claim 7
It is：The condition of detection described in step (c) is：
Chromatographic column：SHIMADZU InertSustain C18 column, 5 μm, 250 × 4.6mm；
Mobile phase：The volume ratio of -0.1% phosphate aqueous solution of methanol, methanol and phosphate aqueous solution is 75:25；
Flow velocity：0.6~1.0mL/min；
Column temperature：25~35 DEG C；
Detection wavelength：515nm；
Sample volume：1~10 μ L.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105203663A (en) * | 2015-09-18 | 2015-12-30 | 西安科技大学 | Rapid detection method for alkannin |
| CN107102084A (en) * | 2017-05-24 | 2017-08-29 | 江苏耐雀生物工程技术有限公司 | A kind of method for separating and detecting of alkannin chiral isomer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105203663A (en) * | 2015-09-18 | 2015-12-30 | 西安科技大学 | Rapid detection method for alkannin |
| CN107102084A (en) * | 2017-05-24 | 2017-08-29 | 江苏耐雀生物工程技术有限公司 | A kind of method for separating and detecting of alkannin chiral isomer |
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