CN101303359B - Method for quantitatively testing halcinonide in cosmetics - Google Patents
Method for quantitatively testing halcinonide in cosmetics Download PDFInfo
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- CN101303359B CN101303359B CN2008100626109A CN200810062610A CN101303359B CN 101303359 B CN101303359 B CN 101303359B CN 2008100626109 A CN2008100626109 A CN 2008100626109A CN 200810062610 A CN200810062610 A CN 200810062610A CN 101303359 B CN101303359 B CN 101303359B
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- halcinonidedcorten
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- atractylenolide
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Abstract
The invention discloses a method for the quantitative detection of halcinonide in cosmetics, which comprises the steps that: 1) halcinonide and butenolide II are added into chromatographic methanol to obtain a reference substance; 2) a butenolide II methanol solution is added into a centrifuge tube and then cosmetics to be detected are added into the centrifuge tube which is dried in a manner of blow; 3) an extraction reagent is added; 4) centrifugation is carried out to obtain all organic phase solutes; 5) the organic phase solutes are dissolved in a mobile phase and a liquid layer is obtained after centrifugation; 6) the reference substance obtained by the step 1) and the liquid layer by the step 5) are carried out high efficiency liquid chromatographic analysis respectively; 7) the content of halcinonide in cosmetics to be detected is equal to the absorption peak area ratio of the halcinonide and the butenolide II in the liquid layer which is multiplied by the concentration of the halcinonide in the reference substance and divided by the absorption peak area ratio of the halcinonide and the butenolide II in the reference substance. The method for halcinonide detection provided by the invention has the advantage of high accuracy.
Description
Technical field
The present invention relates to glucocorticoid in a kind of cosmetics---the halcinonidedcorten quantitative detection method.
Background technology
Halcinonidedcorten (Halcinonide) is a kind of glucocorticoid medicine, and clinical research finds that halcinonidedcorten can effectively be treated the limitation chronic eczema with Calcipotriol associating external application, and total effective rate reaches 88.9%; Halcinonidedcorten and Calcipotriol Combined application have significant curative effect to the treatment psoriasis vulgaris, and psoriasis district severity index (PASI) average rate of change is single with group apparently higher than both.Simultaneously, halcinonidedcorten uses the damage of treatment chemotherapy seepage property separately, has good curative effect.In a word, halcinonidedcorten has good curative effect aspect treatment psoriasis and the clinical common skin diseases.
Cosmetics and daily life are closely related, and along with the raising of China's living standards of the people, cosmetics have become one of requisite consumer goods of people in recent years.2004, China's cosmetics sales volume reached 85,000,000,000 yuans, and increased with annual 15% amplitude.Meanwhile, the security of cosmetics has become the problem that consumers in general pay close attention to day by day.The chemical substance of being added in the cosmetics has potential harm to health.
The glucocorticoid external application can reduce the permeability of capillary, reduces to ooze out and cellular infiltration, has multiple effects such as anti-inflammatory, antiallergy, anti-shock, immunosupress, and clinical practice is extensive.But contact for a long time glucocorticoid be prone to cause thinning of skin, rubescent, itch, degradation spinoff under blood sugar increasing, hypertension, osteoporosis, fetal anomaly, the immunologic function appears.Short time uses the cosmetics that contain glucocorticoid can suppress chafing, recovers skin elasticity, reduces wrinkle or treatment acne, promotes hair growth; But the long-term cosmetics that contain glucocorticoid that use just can cause various spinoffs, produce hormonal dependent, even can cause cancer.Therefore in China's hygienic standards for cosmetics, cosmetics health standard and the European Union's cosmetics rules all clearly the regulation glucocorticoid be the banned substance in the cosmetics.In recent years; Report about in cosmetics, detecting glucocorticoid and generation toxic and side effect emerges in an endless stream, and therefore the glucocorticoid detection method is the important means of hormone toxic and side effect mechanism in the further investigation cosmetics in sensitive, accurate, the reliable cosmetics of foundation.
Existing a kind of method that detects halcinonidedcorten------ultraviolet spectrophotometer method, it has the accurate test result characteristics; There is following weak point:
1. detection sensitivity is low: the detectability of ultraviolet spectrophotometer is generally in the milligram rank;
2. ultraviolet spectrophotometer does not have separation function, and impurity disturbs more serious in the cosmetics.
Summary of the invention
The technical matters that the present invention will solve provides the quantitative detecting method of halcinonidedcorten in a kind of cosmetics, adopts this method to detect halcinonidedcorten and has advantage of high accuracy.
In order to solve the problems of the technologies described above, the present invention provides the quantitative detecting method of halcinonidedcorten in a kind of cosmetics, may further comprise the steps:
1), adding purity is 100% halcinonidedcorten and atractylenolide I in chromatogram methyl alcohol, evenly mixes back one-tenth reference substance; The concentration of halcinonidedcorten in reference substance is 0.1～10 μ g/mL, and the concentration of atractylenolide I in reference substance is 8～12 μ g/mL;
2), adding dries up centrifuge tube as the atractylenolide I methanol solution 0.2mL of internal standard compound in dry zone plug centrifuge tube, in the centrifuge tube after then cosmetics 0.2mL adding to be measured being dried up; The concentration of atractylenolide I in atractylenolide I methanol solution equals the concentration of atractylenolide I in reference substance;
3), in above-mentioned steps 2) add the extraction reagent of 4.5～5.5mL in the centrifuge tube of gained;
4), centrifugal with carrying out with 4000～10000r/min behind the centrifuge tube of the step 3) gained vibration mixing, obtain whole organic phase dissolved matters; And the organic phase dissolved matter that is obtained carried out dried;
5), the organic phase dissolved matter after the above-mentioned dried is dissolved in the acetonitrile of volume ratio=50: 50 and the 0.2mL moving phase that water is formed, after 15000～30000r/min is centrifugal, obtain liquid level;
6), the reference substance of step 1) gained and the liquid level of step 5) gained are carried out efficient liquid phase chromatographic analysis respectively; Testing conditions is following: as moving phase, flow velocity 0.5～1.0mL/min, sample size 10～20 μ L, detection wavelength are that 240nm, chromatogram column temperature are 30 degrees centigrade with the acetonitrile of volume ratio=50: 50 and water; Obtain the absorption peak area ratio of halcinonidedcorten and atractylenolide I in reference substance and the liquid level respectively;
7), obtain the content of halcinonidedcorten in the cosmetics to be measured according to following computing formula:
The absorption peak area ratio of halcinonidedcorten and atractylenolide I in the concentration/reference substance of absorption peak area ratio * halcinonidedcorten in reference substance of halcinonidedcorten and atractylenolide I in the content=liquid level of halcinonidedcorten in the cosmetics to be measured.
Improvement as the quantitative detecting method of halcinonidedcorten in the cosmetics of the present invention: the extraction reagent in the step 3) is ethyl acetate.
Further improvement as the quantitative detecting method of halcinonidedcorten in the cosmetics of the present invention: step 2) in 40～50 ℃ of water-baths nitrogen stream dry up centrifuge tube.
Further improvement as the quantitative detecting method of halcinonidedcorten in the cosmetics of the present invention: in the step 4) centrifugal after; The supernatant liquid of centrifugal back gained is poured in another centrifuge tube; Nitrogen stream dries up this another centrifuge tube in 45 ℃ of water-baths then, the organic phase dissolved matter after the dried.
Further improvement as the quantitative detecting method of halcinonidedcorten in the cosmetics of the present invention: the concentration of atractylenolide I in reference substance is 10 μ g/mL in the step 1), adds the ethyl acetate of 5mL in the step 3).
When cosmetics to be checked are liquid state or paste, adopt method of the present invention can learn the halcinonidedcorten that contains how many μ g in every mL cosmetics to be checked.When cosmetics to be checked are powdery class solid, can draw every cm earlier
3The halcinonidedcorten that contains how many μ g in (being equal to every mL) cosmetics to be checked then according to its proportion, is learnt the halcinonidedcorten that contains how many μ g in every g cosmetics to be checked after the conversion.
Therefore the quantitative detecting method of halcinonidedcorten in the cosmetics of the present invention has adopted high performance liquid chromatography (HPLC) method, can make detectability reach Gamma Magnitude, can satisfy the detection requirement that the hormone of national cosmetic is limited the quantity of fully.And high performance liquid chromatography (HPLC) method has separation and analyzes quantitative function; Can hormone chromatographic peak in the cosmetics and impurity Interference Peaks be separated; Carry out quantitative test, can avoid the interference of the impurity in the cosmetics, thereby guaranteed the correctness of testing result testing result.
In order to prove the correctness of the inventive method, the inventor has been following contrast experiment:
Select a kind of the confirmed relaxed and comfortable liquid cosmetics of not chloride fluorine (for example commercially available pool is beautiful), its chromatogram (the same the present invention of stratographic analysis testing conditions) as shown in Figure 2 for use.In this kind cosmetics, add the halcinonidedcorten of Different Weight, thereby form the testing sample that contains the variable concentrations halcinonidedcorten.Adopt method of the present invention and spectrophotometer method to detect respectively to above-mentioned testing sample; The result is as described in Table 1:
Table 1
| The concentration of halcinonidedcorten in the testing sample (μ g/ml) | The testing result of the inventive method (μ g/ml) | The result of spectrophotometer method gained (μ g/ml) | |
| |
0 | 0 | ND |
| Sample A | 0.2 | 0.19 | ND |
| Sample B | 0.5 | 0.51 | ND |
| Sample C | 0.8 | 0.79 | |
| Sample D | |||
| 1 | 0.99 | ND |
Annotate: the ND in the table representes not detect.
Can be known by table 1: method of the present invention has the advantage that test result is accurate, highly sensitive, inclusion-free disturbs with respect to the spectrophotometer method.
Description of drawings
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is the standard colors spectrogram of halcinonidedcorten and atractylenolide I (interior mark); Wherein: peak 1 is the halcinonidedcorten chromatographic peak, and peak 2 is interior mark atractylenolide I chromatographic peak;
Fig. 2 is the chromatogram of cosmetic base;
Fig. 3 is halcinonidedcorten and atractylenolide I (interior mark) chromatogram in embodiment 1 described a kind of liquid cosmetics; Wherein: peak 1 is the halcinonidedcorten chromatographic peak; Peak 2 is interior mark atractylenolide I chromatographic peak.
Embodiment
The quantitative detecting method of halcinonidedcorten in embodiment 1, a kind of cosmetics, these cosmetics to be measured are the liquid state cosmetics, carry out following steps successively:
1), in chromatogram methyl alcohol, to add purity be 100% halcinonidedcorten and atractylenolide I, evenly mixes the back and become reference substance, halcinonidedcorten and the concentration of atractylenolide I in reference substance are respectively 5 μ g/mL and 10 μ g/mL.
2), at first in dry zone plug centrifuge tube, add atractylenolide I methanol solution 0.2mL as internal standard compound; The concentration of atractylenolide I methanol solution is 10 μ g/mL, contains 10 μ g atractylenolide I in the solution that promptly every mL is made up of chromatogram methyl alcohol and atractylenolide I; Then this centrifuge tube nitrogen stream in 45 ℃ of water-baths is dried up; In this centrifuge tube, add 0.2mL cosmetics to be measured then.
3), according to the ethyl acetate consumption to different with as the extraction ratio of the atractylenolide I of internal standard compound of hormone in the cosmetics, to above-mentioned steps 2) add 5mL ethyl acetate in the centrifuge tube of gained.
4), with above-mentioned steps 3) centrifuge tube of gained carries out following operation:
With its ultrasonic 2min, vortex oscillation 5min carries out centrifugal with 4000～10000r/min behind the mixing; The supernatant liquid of centrifugal back gained is poured in another centrifuge tube, and nitrogen stream dries up this another centrifuge tube in 45 ℃ of water-baths then, the organic phase dissolved matter after the dried, this moment, the organic phase dissolved matter was attached on the tube wall in another centrifuge tube.
5), in above-mentioned steps 4) add the moving phase that 0.2mL is made up of the acetonitrile and the water of volume ratio=50: 50 in another centrifuge tube of gained, above-mentioned organic phase dissolved matter is dissolved in the moving phase; After 15000～30000r/min is centrifugal, obtain liquid level.
6), the reference substance of step 1) gained and the liquid level of step 5) gained are carried out efficient liquid phase chromatographic analysis respectively, testing conditions is following:
With day island proper Tianjin high performance liquid chromatography; Two yuan of high-pressure pumps; Ultraviolet/visible detection device; SHIMADUZ chromatographic column (150mm * 4.6mm inserts particle diameter 5 μ m), with volume ratio be 50: 50 acetonitrile and water as moving phase, flow velocity 0.5～1.0mL/min, sample size 10～20 μ L, detection wavelength are that 240nm, detected temperatures are 30 degrees centigrade.
In the step 1) gained reference substance, the halcinonidedcorten peak area is 199335.078, and atractylenolide I peak area is 209400.906, absorption peak area ratio=0.95193 of its halcinonidedcorten and atractylenolide I.
In the liquid level of the cosmetics to be measured of step 6) gained, halcinonidedcorten peak area 36071.566, atractylenolide I peak area is 183727.141, absorption peak area ratio=0.19633 of its halcinonidedcorten and atractylenolide I.
7), obtain the content of halcinonidedcorten in the cosmetics to be measured according to following computing formula:
The absorption peak area ratio of halcinonidedcorten and atractylenolide I in the concentration/reference substance of absorption peak area ratio * halcinonidedcorten in reference substance of halcinonidedcorten and atractylenolide I in the content=liquid level of halcinonidedcorten in the cosmetics to be measured.
Therefore, content=0.19633 * 5 of halcinonidedcorten (μ g/mL) ÷ 0.95193=1.03 (μ g/mL) in the cosmetics to be measured.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (4)
1. the quantitative detecting method of halcinonidedcorten in the cosmetics is characterized in that may further comprise the steps:
1), adding purity is 100% halcinonidedcorten and atractylenolide I in chromatogram methyl alcohol, evenly mixes back one-tenth reference substance; The concentration of halcinonidedcorten in reference substance is 0.1～10 μ g/mL, and the concentration of atractylenolide I in reference substance is 8～12 μ g/mL;
2), adding dries up centrifuge tube as the atractylenolide I methanol solution 0.2mL of internal standard compound in dry zone plug centrifuge tube, in the centrifuge tube after then cosmetics 0.2mL adding to be measured being dried up; The concentration of atractylenolide I in atractylenolide I methanol solution equals the concentration of atractylenolide I in reference substance;
3), in above-mentioned steps 2) add the extraction reagent of 4.5～5.5mL in the centrifuge tube of gained; Said extraction reagent is ethyl acetate;
4), centrifugal with carrying out with 4000～10000r/min behind the centrifuge tube of the step 3) gained vibration mixing, obtain whole organic phase dissolved matters; And the organic phase dissolved matter that is obtained carried out dried;
5), the organic phase dissolved matter after the above-mentioned dried is dissolved in the acetonitrile of volume ratio=50: 50 and the 0.2mL moving phase that water is formed, after 15000～30000r/min is centrifugal, obtain liquid level;
6), the reference substance of step 1) gained and the liquid level of step 5) gained are carried out efficient liquid phase chromatographic analysis respectively; Testing conditions is following: as moving phase, flow velocity 0.5～1.0mL/min, sample size 10～20 μ L, detection wavelength are that 240nm, chromatogram column temperature are 30 degrees centigrade with the acetonitrile of volume ratio=50: 50 and water; Obtain the absorption peak area ratio of halcinonidedcorten and atractylenolide I in reference substance and the liquid level respectively;
7), obtain the content of halcinonidedcorten in the cosmetics to be measured according to following computing formula:
The absorption peak area ratio of halcinonidedcorten and atractylenolide I in the concentration/reference substance of absorption peak area ratio * halcinonidedcorten in reference substance of halcinonidedcorten and atractylenolide I in the content=liquid level of halcinonidedcorten in the cosmetics to be measured.
2. the quantitative detecting method of halcinonidedcorten in the cosmetics according to claim 1 is characterized in that: said step 2) in 40～50 ℃ of water-baths nitrogen stream dry up centrifuge tube.
3. the quantitative detecting method of halcinonidedcorten in the cosmetics according to claim 2; It is characterized in that: in the said step 4) centrifugal after; The supernatant liquid of centrifugal back gained is poured in another centrifuge tube; Nitrogen stream dries up this another centrifuge tube in 45 ℃ of water-baths then, the organic phase dissolved matter after the dried.
4. the quantitative detecting method of halcinonidedcorten in the cosmetics according to claim 3 is characterized in that: the concentration of atractylenolide I in reference substance is 10 μ g/mL in the said step 1), adds the ethyl acetate of 5mL in the step 3).
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Non-Patent Citations (4)
| Title |
|---|
| 李伟等.HPLC法测定白术中白术内酯II的含量.《现代中药研究与实践》.2005,第19卷(第5期),第35-36页. * |
| 温晓娜.HPLC法测定氟氯霜中的两组分的含量.《天津药学》.1999,第11卷(第2期),第66-67页. * |
| 王学艳等.HPLC法测定牛皮癣乳膏中维A酸和哈西奈德的含量.《中国药房》.2007,第18卷(第22期),第1740-1741页. * |
| 陈頔等.高效液相色谱-串联质谱检测外用制剂中7种激素.《中国药学杂志》.2007,第42卷(第18期),第1431-1433页. * |
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