Method for determining prohibited components in anti-hair loss cosmetic

Abstract

The application discloses a method for determining the content of forbidden components in anti-hair-loss cosmetics, and comprises the following steps: after sample treatment, machine determination is carried out, and an HPLC-MS / MS detection method is adopted, wherein: the liquid chromatography conditions are as follows: a chromatographic column is an Agilent ZORBAX SB-C18 (10 cm*4.6 mm, 3.5 um); a mobile phase A is 0.1% formic acid aqueous solution; a mobile phase B is acetonitrile; a flow rate is 0.6 mL / min; a column temperature is 25 DEG C; and a sample injection amount is 5 uL; and the forbidden components are finasteride and / or fluoroandrosterone. The method for determining the forbidden components in the anti-hair-loss cosmetics has high sensitivity, is simple and easy to operate, and has good repeatability, is a kind of detection method capable of accurately determining the nature and quantity, and has important significance for controlling the quality of the anti-hair-loss cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting chemical substances, in particular to a method for determining the banned components in anti-hair loss cosmetics. BACKGROUND

[0002] Hair loss is a common clinical disease. The main drugs for treating hair loss in modern medicine are androgen blockers, biological response modifiers, and corticosteroids. With the increasing demand for hair care, many products claiming to have hair-nourishing and hair-loss-preventing effects have appeared on the market. These products have the effects of promoting hair growth, producing new hair, and preventing hair loss. Some unscrupulous manufacturers illegally add a large amount of banned substances, such as finasteride and fluorolediol, to these products to produce rapid effects. There is no corresponding detection method for fluorolediol in cosmetics, and there is no report on the simultaneous quantitative analysis of finasteride and fluorolediol by high performance liquid chromatography tandem mass spectrometry. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a method for determining finasteride and fluorolediol in anti-hair loss cosmetics, which has high sensitivity, is simple to operate, and has good repeatability.

[0004] The method for determining the banned components in anti-hair loss cosmetics of the present application comprises the following steps: treating the sample and then determining it on the machine, using HPLC-MS / MS detection method, wherein:

[0005] The liquid chromatography conditions are as follows:

[0006] Chromatographic column: Agilent ZORBAX SB-C18 (10 cm*4.6 mm, 3.5 μm);

[0007] Mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile.

[0008] Flow rate: 0.6 mL / min;

[0009] Column temperature: 25℃;

[0010] Injection volume: 5 μL;

[0011] The banned components are finasteride and / or fluorolediol.

[0012] The method for determining the banned components in anti-hair loss cosmetics of the present application, wherein:

[0013] Table 1 Gradient conditions of mobile phase

[0014]

[0015] The gradient elution procedure in the liquid chromatography condition is shown in Table 1.

[0016] The method for determining the forbidden components in the anti-hair loss cosmetic according to the present application, wherein:

[0017] The mass spectrometry condition is as follows:

[0018] Ion source: electrospray ion source ESI;

[0019] Scan mode: finasteride is positive ion scan; fluridil is negative ion scan;

[0020] Ion source parameters:

[0021] Gas curtain: 35.0 psi;

[0022] Collision gas: 10 psi;

[0023] Ion spray voltage: 4500V;

[0024] Ion source temperature: 550℃;

[0025] Atomization gas: 60 psi;

[0026] Auxiliary gas: 65 psi;

[0027] Detection mode: multiple reaction monitoring.

[0028] The method for determining the forbidden components in the anti-hair loss cosmetic according to the present application, wherein:

[0029] The mass spectrometry parameters of the finasteride and fluridil are shown in Table 2:

[0030] Table 2 Characteristic ions and mass spectrometry parameters of finasteride and fluridil

[0031]

[0032] * is a quantitative ion.

[0033] The method for determining the forbidden components in the anti-hair loss cosmetic according to the present application, wherein:

[0034] The sample processing method comprises the following steps:

[0035] 1g of the cosmetic sample is weighed in a 10mL cuvette, a solvent of acetonitrile and methanol in a volume ratio of 95:5 is added, the volume is made up to 10mL, vortexed for 2min, then placed in an ultrasonic extractor for extraction for 20min, after taking out from the ultrasonic extractor, vortexed for 2min to mix, then centrifuged at 6000r / min for 8min; the supernatant is taken through a 0.22μm filter membrane into a sample vial, and then the sample is injected into the machine for analysis

[0036] The method for determining the banned components in the anti-hair loss cosmetic of the present application is different from the prior art in that:

[0037] The method for determining the banned components in the anti-hair loss cosmetic of the present application adopts HPLC-MS / MS detection technology, establishes a method for simultaneously detecting finasteride and fluorouracil in the anti-hair loss cosmetic, and can also be used for separately detecting finasteride or fluorouracil, the method is high in sensitivity, simple in operation, good in repeatability, is a detection method capable of accurately determining the nature and quantity, and has important significance for controlling the quality and safety of the anti-hair loss cosmetic.

[0038] The method for determining the banned components in the anti-hair loss cosmetic of the present application will be further described below in combination with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 For comparison of chromatograms of different mobile phases in the present application;

[0040] Figure 2 For comparison of chromatograms of different flow rates in the present application;

[0041] Figure 3 For comparison of chromatograms of different chromatographic columns in the present application;

[0042] Figure 4 For the total chromatogram of finasteride and fluorouracil obtained by the method of the present application;

[0043] Figure 5 For the MRM diagram of finasteride obtained by the method of the present application;

[0044] Figure 6 For the MRM diagram of fluorouracil obtained by the method of the present application. DETAILED DESCRIPTION

[0045] I. Materials and reagents

[0046] Finasteride and fluorouracil standard (Germany Dr Ehrenstorfer Company, purity ≥ 95%); acetonitrile, methanol, formic acid (chromatographic pure, USA Fisher Company); other reagents are all analytical pure.

[0047] 10mL centrifuge tube with plug, Yue Xiu Technology (Shanghai) Co., Ltd.

[0048] II. Instruments and equipment

[0049] AB SCIEX QTRAP 5500 high-performance liquid chromatograph tandem mass spectrometer; German SCILOGEX vortex shaker; Hitachi CF-16RN high-speed refrigerated centrifuge; American Mettler analytical balance; American Milli-Q ultrapure water generator; Chinese Sume ultrasonic cleaner.

[0050] III. Preparation of standard stock solution

[0051] Accurately weigh 10 mg (precise to 0.0001 g) of finasteride and fluoroandrosterone standard into two 10 mL volumetric flasks, respectively. After ultrasonic dissolution with methanol, make up to the 10 mL mark, shake well, and prepare the standard stock solution with a concentration of 1000 mg / L. Store in the refrigerator (4°C) and can be stored for 3 months.

[0052] Mixed standard stock solution: Transfer 10 μL of finasteride stock solution and 100 μL of fluoroandrosterone stock solution into the same 10 mL volumetric flask, respectively. After ultrasonic dissolution with methanol, make up to the 10 mL mark, shake well, and prepare the mixed standard stock solution with a concentration of 1 mg / L of finasteride and 10 mg / L of fluoroandrosterone. Store in the refrigerator (4°C) and can be stored for 1 month.

[0053] IV. Instrument conditions

[0054] 1. Liquid chromatography conditions

[0055] a) Column: Agilent ZORBAX SB-C18 (10 cm*4.6 mm, 3.5 μm);

[0056] b) Mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile. Gradient elution program: see Table 3;

[0057] Table 3 Gradient conditions of mobile phase

[0058]

[0059] c) Flow rate: 0.6 mL / min;

[0060] d) Column temperature: 25°C;

[0061] e) Injection volume: 5 μL.

[0062] 2. Mass spectrometry conditions

[0063] a) Ion source: electrospray ion source (ESI);

[0064] b) Scan mode: positive ion scan for finasteride; negative ion scan for fluoroandrosterone;

[0065] c) Ion source parameters: curtain gas (CUR) 35.0 psi; collision gas (CAD) 10 psi

[0066] Ion spray voltage (IS) 4500 V

[0067] Ion source temperature (TEM) 550°C

[0068] Nebulizer gas (GS1) 60 psi; Auxiliary gas (GS2) 65 psi

[0069] d) Detection mode: Multiple reaction monitoring (MRM);

[0070] e) The mass spectrometry parameters of finasteride and furazolidone are shown in Table 4.

[0071] Table 4 Characteristic ions and mass spectrometry parameters of finasteride and furazolidone

[0072]

[0073] * Quantitative ion.

[0074] 3. Sample processing method

[0075] Weigh 1 g (accurate to 0.001 g) of the cosmetic sample into a 10 mL cuvette, add acetonitrile:methanol (95:5), dilute to 10 mL, vortex for 2 min, then put it into an ultrasonic extractor for extraction for 20 min, after taking it out of the ultrasonic extractor, vortex for 2 min to mix, then centrifuge at 6000 r / min for 8 min.

[0076] Take the supernatant through a 0.22 μm filter membrane into a sample vial, and inject it into the machine for analysis.

[0077] Five, results and discussion

[0078] 1. Optimization of liquid phase conditions

[0079] 1.1 Selection of mobile phase

[0080] This experiment investigated the influence of methanol-0.1% formic acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution, methanol-5 mmol / L ammonium acetate aqueous solution and acetonitrile-5 mmol / L ammonium acetate aqueous solution mixed solution as the mobile phase on the peak shape and sensitivity of finasteride and furazolidone. Figure 1 The results showed that when the mobile phase was acetonitrile-0.1% formic acid aqueous solution, the peak shape of finasteride and furazolidone could be improved, and the sensitivity was also improved, and finally acetonitrile-0.1% formic acid aqueous solution was determined as the mobile phase.

[0081] 1.2 Selection of flow rate

[0082] For liquid chromatography mass spectrometry analysis, the size of the flow rate is very important, this invention investigated the mass spectrometry response signal and chromatographic separation effect of finasteride and furazolidone in the range of 0.4-0.8 mL / min. Figure 2). Studies show that at a flow rate of 0.6 mL / min, the separation effect of finasteride and fluorogil, analysis time, and column pressure of the chromatographic column are ideal, finasteride and fluorogil have smaller peak broadening, thereby realizing the optimal conditions for chromatographic separation and mass spectrometric analysis of the target extract.

[0083] 1.3 Selection of chromatographic column

[0084] The present application respectively investigates four chromatographic columns with different selectivity, mainly including ATLANTIS T3 chromatographic column (strong retention ability for polar compounds, stable performance in water mobile phase), C18 chromatographic column (filler is octadecyl bonded silica, used for separating substances with larger polarity), C8 chromatographic column (octane silane bonded silica gel) and HILIC chromatographic column (used for improving strong polar compounds with poor retention in reverse phase chromatography). The results show that finasteride and fluorogil obtain the most ideal separation effect and better peak shape on Agilent ZORBAX SB-C18 (10 cm*4.6 mm, 3.5 μm) chromatographic column Figure 3 ).

[0085] 2. Optimization of mass spectrometry conditions

[0086] According to the structural characteristics of finasteride and fluorogil, finasteride is positive ion scanning; fluorogil is negative ion scanning. First, one-stage spectrum scanning is performed on each substance, the parent ion is selected from the one-stage mass spectrum, the fragment ion information is obtained by breaking the parent ion, and then two fragment ions with higher response values are selected as daughter ions to form an ion pair with the parent ion. The ion source temperature, residence time and collision chamber outlet voltage are optimized, so that the ionization efficiency of each substance in the sample reaches the best. The MRM diagram of the quantitative ions of finasteride and fluorogil is shown in Figure 5 and Figure 6 , and the total ion flow diagram is shown in Figure 4 .

[0087] 3. Optimization of sample pretreatment method

[0088] Since different extraction solvents and extraction time have certain influence on the recovery rate of target substances, several different extraction solvents such as acetonitrile, methanol, acetonitrile: methanol (v / v=95:5), 0.2% formic acid acetonitrile solution, 0.2% formic acid methanol solution, etc. were used to investigate the influence of solvents on the extraction effect of target substances. In addition, the influence of different extraction time (10, 15, 20, 25, 30 min) on the recovery rate of target substances was investigated (Table 5 and Table 6). The results showed that when acetonitrile: methanol (95:5) was used as the extraction solvent, the extraction recovery rate of finasteride and fluoroandrosterone was the highest (Table 5), and the interference with mass spectrometric detection was very small; when the extraction time was 20 min, the recovery rate of the target substance was stable, and the recovery rate of 81.7% to 104.1% was obtained (Table 6). Therefore, the optimal extraction method was determined to be acetonitrile: methanol (v / v=95:5) extraction for 20 min.

[0089] Table 5 Recovery rate results when the extraction solvent is different

[0090]

[0091] Table 6 Recovery rate results when the extraction time is different

[0092]

[0093] 4. Linear range, detection limit and quantification limit of the method

[0094] 4.1 Linear regression equation, detection limit and quantification limit

[0095] The standard working solution was obtained by diluting the standard stock solution of finasteride and fluoroandrosterone step by step, and was analyzed by sampling according to the determination conditions of 2.4. The standard curve was drawn with the peak area (Y axis) of finasteride and fluoroandrosterone against the corresponding concentration (X axis), and the correlation coefficient was above 0.999. In addition, the linear regression equation and the linear range were obtained, as shown in Table 7. The blank sample was added with standard, and the detection limit of the method was obtained by 3 times the signal-to-noise ratio; and the quantification limit was obtained by 10 times the signal-to-noise ratio.

[0096] Table 7 Linear range, regression equation, detection limit and quantification limit of finasteride and fluoroandrosterone

[0097]

[0098] 4.2 Spiked recovery rate and precision

[0099] The method adds standard solutions with different concentrations to blank samples such as water, emulsion and cream, carries out addition recovery and precision experiment, and carries out sample determination by using HPLC-MS / MS. The average recovery rate (6 times of parallel determination for each addition concentration) in the range of low, medium and high addition levels is between 81.5% and 106.5%, and the recovery rate result is shown in Table 8.

[0100] Table 8 Recovery rate and precision experiment result of finasteride and fluocinolide (n = 6)

[0101]

[0102] Six, determination of actual samples

[0103] Water, milk and cream (total 20 anti-hair loss samples) are purchased from different supermarkets, and the method is used for determination, and it is found that there is one case of label inconsistency. The detection results of other products are all not detected.

[0104] Seven, conclusion

[0105] The application establishes a liquid chromatography-tandem mass spectrometry analysis method for simultaneously determining the drug concentration of finasteride and fluocinolide in anti-hair loss cosmetics, realizes the characteristics of high sensitivity and good recovery rate, can meet the demand of content determination of finasteride and fluocinolide in anti-hair loss cosmetics, provides a new method for quality and safety control of anti-hair loss cosmetics, and has important significance for quality and safety control of cosmetics.

[0106] The above-described embodiments are merely preferred embodiments of the application, and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements of the technical solutions of the application made by those skilled in the art shall fall within the protection scope of the claims of the application.

Claims

1. A method for determining a prohibited component in an anti-hair loss cosmetic product, characterized by: The method comprises the following steps: after the sample is treated, the sample is determined by using a HPLC-MS / MS detection method, and the extraction solvent is a solvent with a volume ratio of 95:5 of acetonitrile and methanol. The liquid chromatography conditions are as follows: Column: Agilent ZORBAX SB-C18, 10 cm 4.6 mm, 3.5 μm; Mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; The gradient elution program is as follows: Flow rate: 0.6 mL / min; Column temperature: 25 DEG C; The banned components are finasteride and fluoro.

2. The method for determining the prohibited component in the anti-hair loss cosmetic according to claim 1, characterized in that: The liquid chromatography conditions further comprise: injection volume: 5 mu L.

3. The method for determining the banned components in the anti-hair loss cosmetic product according to claim 1, characterized in that: The mass spectrometry conditions are as follows: Ion source: electrospray ion source ESI; Scan mode: positive ion scan for finasteride and negative ion scan for fluoro; Ion source parameters: Gas curtain gas: 35.0 psi; Collision gas: 10 psi; Ion spray voltage: 4500 V; Ion source temperature: 550 DEG C; Atomization gas: 60 psi; Auxiliary gas: 65 psi; Detection mode: multiple reaction monitoring.

4. The method for determining the presence of a prohibited ingredient in an anti-hair loss cosmetic product according to claim 3, characterized in that: The mass spectrometry parameters of the finasteride and fluoro are as follows: To quantify ions.

5. The method for determining the prohibited component in the anti-hair loss cosmetic according to claim 1, characterized in that: The sample treatment method comprises the following steps: 1 g of the cosmetic sample is weighed in a 10 mL colorimetric tube, a solvent with a volume ratio of 95:5 of acetonitrile and methanol is added, the volume is adjusted to 10 mL, vortexed for 2 min, then placed in an ultrasonic extractor for extraction for 20 min, after being taken out from the ultrasonic extractor, vortexed for 2 min, mixed uniformly, and then centrifuged at 6000 r / min for 8 min; the supernatant is taken, filtered through a 0.22 mu m filter membrane into a sample injection vial, and then injected into a machine for analysis.

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