CN101169396A - Cosmetic product betamethasone high efficiency liquid chromatography detection method - Google Patents
Cosmetic product betamethasone high efficiency liquid chromatography detection method Download PDFInfo
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- CN101169396A CN101169396A CNA2007100279236A CN200710027923A CN101169396A CN 101169396 A CN101169396 A CN 101169396A CN A2007100279236 A CNA2007100279236 A CN A2007100279236A CN 200710027923 A CN200710027923 A CN 200710027923A CN 101169396 A CN101169396 A CN 101169396A
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Abstract
The invention provides an analysis method of detecting the betamethasone in cosmetic by use of high efficiency liquid chromatographic instrument, and the invention is characterized in that octadecylsilane chemically bonded silica is adopted as the C18 column or the C8 column of filler, and an ultraviolet detector or a diode array detector are used to detect at the position of 190 to 380 nm; the mobile phase adopts methanol-water mixed solution or acetonitrile-water mixed solution; the sample is extracted by methanol-hydrochloric acid mixed solution or acetonitrile-hydrochloric acid mixed solution, extracted by ultrasound for around 20 minutes, and undergone with an ice bath, and then is centrifuged in 12000rpm/min for 5 minutes. The clear liquid is prepared, which is, if necessary, filtered by 0.45Mu meters filtering membrane for using. The qualitative analysis is conducted according to the sample retention time and the spectrogram, and the quantitative analysis is conducted according to the peak area. The invention has the advantages of high solution and sensitivity, and perfect reproducibility and selectivity as well as simple sample pre-treating method, thereby being applicable to microanalysis of betamethasone in cosmetic.
Description
Technical field
The present invention is a new technology of utilizing high performance liquid chromatograph to detect forbidding composition one betamethasone (Betamethasone) in the cosmetics, is particularly useful for the qualitative and quantitative test of betamethasone in the cosmetics.
Background technology
Betamethasone belongs to adrenal cortex hormones drug, has effect antibiotic, anti-inflammatory.Versions in 2002 " cosmetics health standard " regulation of health ministry promulgation must not be added betamethasone in the cosmetics.Betamethasone is because the bactericidal action of its broad-spectrum high efficacy, is added to the anti-acne series products to reach anti-inflammatory, anti-acne, to remove the purpose of mite and anti-acne.But the bad reaction of this medicine is more, and the effect of Chu sodium is arranged, and is stronger to the inhibiting effect of growth; Can also cause the various bad reactions that corticosteroid hormone causes, as the unusual and Water-Electrolyte disorder of muscle skeleton, intestines and stomach, skin, nervous system, internal system etc.In view of above these bad reactions, the long-term cosmetics or this kind of the skin contact medicine that are added with betamethasone of using can have a strong impact on health.
The bibliographical information that detects about betamethasone mostly is at the detection method of betamethasone in betamethasone raw material and the medicine at present, mainly contains polarimetry, spectrophotometric method, high performance liquid chromatography etc.The polarimetry influence factor is more, needs the amount of standard reference material and sample more, is not suitable for the analysis of micro-betamethasone; The spectrophotometric method complex operation, in the sample preparation process loss more, and detection limit is higher, repeatability is relatively poor; Efficient liquid-phase chromatography method is to detect betamethasone method commonly used in recent years, but also only be confined to the detection of content in bulk drug and the various medicine preparation, because the medicine component is less, matrix is simple, disturbing factor is less, and it is little that the sample pretreatment simple target detects the thing loss, so the content of betamethasone detects easily in the medicine.And the detection country of the betamethasone content in the cosmetics, feed, meat does not also have to formulate relevant examination criteria and method, and up to the present in the cosmetics detection of betamethasone not have the bibliographical information of being correlated with yet.Different with medicine, cosmetics are of a great variety multiple formulations such as cream, frost, breast, powder, water, gel, gel, and matrix complexity, the physicochemical property difference of each composition is bigger, disturbing factor is more, especially betamethasone adds in the cosmetics as cosmetics forbidding composition, and its content is lower, is difficult to detect accurately the betamethasone composition of trace in the cosmetics with above-mentioned polarimetry, spectrophotometric method.Therefore, at the deficiency on the existing betamethasone detection technique, we have designed a kind of method that detects betamethasone in the cosmetics with efficient liquid-phase chromatography method, this method is simple to operate, specificity is good, and is highly sensitive, and the trace detection of betamethasone in the suitable cosmetics.
Summary of the invention
The purpose of this invention is to provide a kind of analytical approach that detects betamethasone in the cosmetics, this technology utilizes high performance liquid chromatograph not only can carry out qualitative analysis accurately to the betamethasone in the cosmetics, but also can carry out the quantitative test of trace.
The physicochemical property that the present invention is directed to betamethasone is extracted betamethasone in the cosmetics with certain density methyl alcohol-hydrochloric acid mixed solution or acetonitrile-hydrochloric acid mixed solution, then with extracting liquid filtering, the filtering solid impurity, clarify until filtrate, get machine on the sample liquid for preparing, carry out wash-out with methanol-water solution or acetonitrile-aqueous solution and separate.The main configuration of used high performance liquid chromatograph comprises that octadecylsilane chemically bonded silica is C18 post or C8 post, diode array detector or the UV-detector of filling agent.
The main analytical procedure of the inventive method is as follows:
1 sample pretreatment
Take by weighing a certain amount of cosmetics (breast, cream, frost or aqua) in the 10ml graduated centrifuge tube, add extract about 6ml (methyl alcohol: 0.01～1.00mol/ml hydrochloric acid=20～80%: 80～20% or acetonitrile: 0.01～1.00mol/ml hydrochloric acid=20～80%: 80～20%), the vortex mixing fully disperses matrix, be settled to scale, ultrasonic Extraction 15-30min again, extract is transferred to ice bath 30min separates out matrix in the Eppendorf pipe, the centrifugal 5min of 12000rpm/min if necessary can be again with 0.45 μ m membrane filtration.
2 chromatographic conditions
(1) chromatographic column: with the octadecylsilane chemically bonded silica is the reverse-phase chromatographic column C18 or the C8 post of filling agent;
(2) detecting device: diode array detector or UV-detector;
(3) detect wavelength: select for use the 190-380nm wavelength as detecting wavelength;
(4) moving phase: adopt methyl alcohol: water=20～80%: 80～20% or acetonitrile: water=20～80%: 80～20% mixed liquors are as moving phase, the about 20min of balance chromatographic column makes baseline steady, and retention time is fit under this condition, the peak shape symmetry, degree of separation good (R＞1.5).
(5) flow velocity: 0.5～1.5ml/min;
(6) column temperature: 15～35 ℃.
3 density calculating methods
Be mixed with the standard solution of certain gradient concentration with the betamethasone standard substance, get 20 μ l sample introductions,, do linear regression with peak area-concentration by external standard method, draw equation of linear regression, utilize the content of betamethasone in the calculated by peak area sample of betamethasone in this equation and the sample that records.
4 sample detection
(1) carries out sample pretreatment according to the method in 1, get the sample 20 μ l sample introductions of handling well; And then get the betamethasone standard solution 20 μ l sample introductions of debita spissitudo.
(2) detect according to the testing conditions in 2, obtain the chromatogram of sample and the chromatogram of betamethasone standard items respectively; The chromatogram of sample and the chromatogram of standard items are compared, the chromatographic peak of close with the standard items retention time among the sample chromatogram figure (error range: standard items retention time ± 0.5 minute) is corresponding betamethasone composition, accuracy in order to ensure testing result, can carry out spectral analysis (spectral analysis is only applicable to diode array detector) to the preliminary chromatographic peak of determining, if, can further determine the betamethasone composition in the sample with the maximum absorption wavelength and the spectrogram feature similarity of standard items.
(3) chromatographic peak area of betamethasone in the sample is brought in the equation of linear regression in 3 into the concentration of betamethasone in the calculation sample.
Because cosmetics are of a great variety, matrix is complicated, can not get rid of the possibility that interference component and betamethasone go out the peak at one time fully.Adopt diode array detector can obtain the uv absorption spectra of betamethasone,,, can further confirm by maximum absorption wavelength and the spectrogram feature of analyzing the two with the spectrogram contrast of standard items.
Analytical approach of the present invention is accurate, easy, quick, and is highly sensitive, and selectivity, good reproducibility are applicable to any cream creme, emulsion, and the cosmetics of various formulations such as aqua.
Description of drawings
Fig. 1 sample chromatogram figure
Fig. 2 betamethasone standard items chromatogram
Fig. 3 sample spectra figure
Fig. 4 betamethasone standard items spectrogram
Fig. 5 betamethasone canonical plotting
Embodiment
The invention will be further elaborated below in conjunction with concrete embodiment, but do not limit the present invention:
1 material
1.1 instrument
Tianjin, island auto injection highly effective liquid phase chromatographic system: Hypersil-ODS (150mm * 4.6mm, 5 μ m, post E1720574) chromatographic column, the SPD-M20A ultraviolet-visible detector, the SPD-M20A diode array detector, LC-20AB binary geopressure gradient pump, SIL-20A automatic sample handling system, CTO-20A column oven and LCSolution chromatographic work station.
1.2 reagent and sample
Absolute methanol (chromatographically pure) is a Merck company product; Hydrochloric acid (top grade is pure) is Tianjin Ke Miou chemical reagent work product; Ultrapure water is produced by Milli-Qsystem.
The betamethasone standard items are purchased in Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and lot number is 100118-200403.
For test agent is quick pox-removing face mask.
1.3 standard items storing solution
Precision takes by weighing 100.00mg betamethasone standard items and places the 50ml volumetric flask, adds the dissolving of a little absolute methanol, and with methanol constant volume to scale, shake up, the titer that is mixed with concentration and is 2mg/ml is deposited standby in 4 ℃ of refrigerators as storing solution.
2 methods
2.1 chromatographic condition
Chromatographic column: Shim-pack VP-ODS (150mm * 4.6mm, 5 μ m)
Detecting device: SPD-M20A diode array detector
Moving phase: adopt methyl alcohol: water=60%: 40% mixed liquor uses the moving phase peak symmetry of this ratio as moving phase, and degree of separation is good.
Detect wavelength: 241nm.Through the 190-400nm length scanning, betamethasone has absorption maximum near 241nm, so select 241nm as detecting wavelength.
Flow velocity: 1ml/min
Column temperature: 25 ℃
2.2 sample pretreatment
The cosmetics (pox-removing face mask fast) that take by weighing 1.00g are in the 10ml graduated centrifuge tube, add the 5ml absolute methanol, the vortex mixing fully disperses matrix, ultrasonic Extraction 20min again, extract is transferred to ice bath 30min in the Eppendorf pipe, the centrifugal 5min of 12000rpm/min uses 0.45 μ m membrane filtration again, makes for test agent solution.
2.3 sample detection
Get 20 μ l test liquid sample introductions, utilize the chromatographic condition in 2.1 to detect, obtain the chromatogram (Fig. 1) and the spectrogram (Fig. 3) of sample; The betamethasone standard items storing solution of getting again in 1.3 dilutes most 60 μ g/ml concentration, and 20 μ l sample introductions under the same testing conditions, obtain the chromatogram (Fig. 2) and the spectrogram (Fig. 4) of betamethasone standard items.
2.4 typical curve
The betamethasone storing solution is diluted to the standard series of following gradient concentration with absolute methanol: 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 60 μ g/ml, 80 μ g/ml, 100 μ g/ml, get the standard solution 20 μ l sample introductions of each concentration, obtain the peak area (seeing Table 1) of each concentration, make peak area-concentration standard curve (see figure 5), obtain its equation of linear regression (finishing evaluation work) by the high performance liquid chromatography workstation.Regression equation: A (area)=78824C-6600 R
2=0.9999
Table 1 betamethasone typical curve each point concentration and peak area
3 interpretations of result
By the chromatogram (Fig. 2) of betamethasone standard items as can be seen, the retention time of utilizing chromatogram testing conditions in 2.1 to obtain the betamethasone standard items is 10.584min, the chromatogram (Fig. 1) of sample and the chromatogram (Fig. 2) of standard items are compared, as seen there is the retention time 10.584min of a chromatographic peak and betamethasone standard items chromatographic peak close at the 10.575min place among the sample chromatogram figure, error is within ± 0.5 minute scope, therefore can determine tentatively that the chromatographic peak at 10.575min place among the sample chromatogram figure is a betamethasone, this peak area is 955842.2mm
2The spectrogram (Fig. 3) at peak, 10.575min place among the sample chromatogram figure and the spectrogram (Fig. 4) of betamethasone standard items are compared, can find the close and spectrogram feature similarity of maximum absorption wavelength of the spectrogram of the spectrogram of sample and standard items, can determine further that retention time in the sample is that the component of 10.575min is a betamethasone.
Chromatographic peak area 1959241.2mm with betamethasone in the sample
2In the substitution equation of linear regression, the content that can obtain betamethasone in the sample preparation liquid is 24.94 μ g/ml (finishing evaluation work by the high performance liquid chromatography workstation), and the content that can get betamethasone in the quick pox-removing face mask sample as calculated is 0.2494mg/kg.
This invention has high-resolution, high sensitivity, reappearance, selective good and simple characteristics of sample pretreating method, in the suitable cosmetics doubly The qualitative and quantitative analysis of Ta Misong.
The present invention has found a kind of analytical method of utilizing high performance liquid chromatograph to detect betamethasone in the cosmetics with great many of experiments and theory analysis, Filled up the research blank of betamethasone liquid-phase chromatographic analysis aspect in the cosmetics.
Claims (8)
1. the high-efficiency liquid chromatography method for detecting of betamethasone in the cosmetics, it is characterized in that adopting UV-detector or diode array detector, with the external standard standard measure, adopting octadecylsilane chemically bonded silica is the C18 post or the C8 post of filling agent, with methanol-water mixture or acetonitrile-water mixed liquor is moving phase, adopts the detection wavelength of 190-380nm, adopts methyl alcohol-hydrochloric acid mixed solution or acetonitrile-hydrochloric acid mixed solution sample dissolution, ultrasonic extraction, 0.45 μ m membrane filtration.
2. the high-efficiency liquid chromatography method for detecting of betamethasone is characterized in that adopting UV-detector or diode array detector in the cosmetics according to claim 1.
3. the high-efficiency liquid chromatography method for detecting of betamethasone in the cosmetics according to claim 1, it is characterized in that adopting octadecylsilane chemically bonded silica is the C18 post or the C8 post of filling agent.
4. the high-efficiency liquid chromatography method for detecting of betamethasone in the cosmetics according to claim 1, it is characterized in that moving phase adopts methyl alcohol: water=20～80%: 80～20% mixed liquor or acetonitrile: water=20～80%: 80～20% mixed liquor, optimal flow are chosen as acetonitrile mutually: water=60%: 40%.
5. the high-efficiency liquid chromatography method for detecting of betamethasone in the cosmetics according to claim 1 is characterized in that adopting the 190-380nm wavelength to detect, and the optimum detection wavelength is 241nm.
6. the high-efficiency liquid chromatography method for detecting of betamethasone in the cosmetics according to claim 1 is characterized in that used flow velocity is 0.5-1.5ml/min, and column temperature is 20-30 ℃, and sampling volume is 20 μ l; Wherein optimum flow rate is 1.00ml/min, and optimum column temperature is 25 ℃.
7. the high-efficiency liquid chromatography method for detecting of betamethasone in the cosmetics according to claim 1,80～20%) or acetonitrile-0.01～1.00mol/ml hydrochloric acid mixed solution (20～80%: 80～20%) dissolution extraction it is characterized in that sample pretreatment adopts methyl alcohol-0.01～1.00mol/ml hydrochloric acid mixed solution (20～80%:, ultrasonic Extraction 15-30min, ice bath 30min, the centrifugal 5min of 12000rpm/min again, supernatant is with 0.45 μ m membrane filtration; Wherein optimization process liquid is acetonitrile-0.1mol/ml hydrochloric acid mixed solution, and its best proportioning is 1: 1.
8. the high-efficiency liquid chromatography method for detecting of betamethasone in the cosmetics according to claim 1 is characterized in that carrying out qualitative analysis according to its retention time and spectrogram behind the sample analysis, utilizes external standard method to carry out quantitative test according to its peak area.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102313687A (en) * | 2011-07-29 | 2012-01-11 | 岳中瑾 | Method for detecting cream preparation for treating phimosis |
| CN104749270A (en) * | 2013-12-27 | 2015-07-01 | 内蒙古出入境检验检疫局检验检疫技术中心 | Method for simultaneously detecting two glucocorticoid isomerides in animal-derived food |
| CN108267525A (en) * | 2016-12-30 | 2018-07-10 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of glucocorticoid in dairy products |
-
2007
- 2007-05-10 CN CNA2007100279236A patent/CN101169396A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102313687A (en) * | 2011-07-29 | 2012-01-11 | 岳中瑾 | Method for detecting cream preparation for treating phimosis |
| CN104749270A (en) * | 2013-12-27 | 2015-07-01 | 内蒙古出入境检验检疫局检验检疫技术中心 | Method for simultaneously detecting two glucocorticoid isomerides in animal-derived food |
| CN104749270B (en) * | 2013-12-27 | 2017-09-12 | 内蒙古出入境检验检疫局检验检疫技术中心 | Detection method while two pairs of glucocorticoid isomers in animal-derived food |
| CN108267525A (en) * | 2016-12-30 | 2018-07-10 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of glucocorticoid in dairy products |
| CN108267525B (en) * | 2016-12-30 | 2021-04-13 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting glucocorticoid in dairy product |
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Open date: 20080430 |