Method for simultaneously detecting diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester in cosmetics

Abstract

The invention discloses a method for simultaneously detecting diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester in cosmetics. According to the method provided by the invention, four compounds such as diaminopyrimidine oxide in cosmetics can be rapidly, completely and accurately extracted; the method for simultaneously determining the four compounds such as diaminopyrimidine oxide in the cosmetics by adopting the high performance liquid chromatography is simple, convenient, efficient and easy to implement. By adopting the method, the sample can be directly measured in the high performance liquid chromatograph without derivatization after being extracted by the extraction solvent, the method is simple and convenient, easy to operate, high in universality, good in reproducibility, high in recovery rate and accurate in detection result, and the quality safety supervision level of cosmetics can be improved; important technical support can be provided for cosmetic industry supervision, and the requirement for cosmetic quality safety supervision is met.

Description

Technical Field

[0001] The present application relates to the field of analysis and detection, and in particular to a method for simultaneously detecting diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester in cosmetics. Background Art

[0002] Diaminopyrimidine oxide (2,4diaminopyrimidine 3oxide) and pyrrolidine diaminopyrimidine oxide (Kopyrrol) are functional raw materials for cosmetics that prevent hair loss and promote hair growth. In cosmetics, diaminopyrimidine oxide can effectively inhibit hair thinning or induce and stimulate hair growth, and can be used to treat baldness, alopecia areata, etc. Pyrrolidine diaminopyrimidine oxide (Kopyrrol) is a new type of hair growth promoter that can effectively promote the proliferation of hair papilla cells. Compared with minoxidil, it has effects such as preventing hair loss and revitalizing hair follicles. At present, pyrrolidine diaminopyrimidine and diaminopyrimidine oxide are both included in the "List of Used Cosmetic Ingredients" (2021 edition), and are allowed to be added to cosmetics as raw materials.

[0003] Metformin is a drug for the treatment of stage 2 diabetes. With the continuous research on metformin, it was found that topical metformin can induce autophagy and hair regeneration. Isopropylcloprostenate is a type of 5α-reductase inhibitor. It can promote hair growth, especially eyelashes, by inhibiting the activity of 5α-reductase. However, long-term use of isopropylcloprostenate can irritate the eyes, cause iris deepening, iritis, etc., and long-term use can even cause blindness. Metformin and isopropylcloprostenate are currently not allowed to be used in cosmetics. However, due to the hair growth promoting effects of metformin and isopropylcloprostenate, some unscrupulous cosmetics merchants will illegally add the above two compounds to cosmetics in order to achieve the effect of promoting hair or eyelash growth.

[0004] At present, the detection methods for metformin are mostly concentrated in the fields of medicines and biological products, and the analytical techniques used are mainly high-performance liquid chromatography, liquid chromatography-mass spectrometry and thin-layer chromatography. There are few reports in the literature on the determination method of metformin in cosmetics. There are few reports in the literature on the determination method of cloprostenol isopropyl ester in cosmetics. There are also reports in the literature that high-performance liquid chromatography is used to determine diaminopyrimidine oxide in cosmetics alone, but there is no report in the literature on the determination method of pyrrolidine diaminopyrimidine oxide in cosmetics.

[0005] In summary, there are few reports on the detection methods of anti-hair loss drugs in cosmetics. There is no literature report on the simultaneous determination of four anti-hair loss drugs in cosmetics, including diaminopyrimidine oxide, pyrrolidinopyrimidine oxide, metformin and cloprostenol isopropyl ester, by high performance liquid chromatography. In addition, due to the complexity of the cosmetic matrix, it is difficult to accurately measure these anti-hair loss substances by referring to the determination methods in the field of chemical drugs. Summary of the invention

[0006] The purpose of the present application is to provide a method for simultaneously detecting diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester in cosmetics.

[0007] In order to solve the above technical problems, the first aspect of the present application provides a method for simultaneously detecting diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester in cosmetics, the method comprising the steps of:

[0008] Liquid chromatography is used to detect whether the cosmetics to be tested contain diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin (or metformin hydrochloride) and cloprostenol isopropyl ester; wherein the chromatographic conditions of the liquid chromatography include: using an aqueous solution of phosphoric acid as the aqueous phase and using methanol as the organic phase for gradient elution, and the procedure of the gradient elution is as follows:

[0009] Time (min) Water phase (v / v%) Organic phase (v / v%) 0 90 10 20 10 90 30 10 90 30.01 90 10 35 90 10 .

[0010] In one embodiment, the mass concentration of phosphoric acid in the aqueous solution of phosphoric acid is 85%.

[0011] In one embodiment, the chromatographic conditions further comprise:

[0012] Chromatographic column: C18 column (preferably AQ-C18 column);

[0013] Column temperature: 25-35°C (e.g. 30°C);

[0014] Flow rate: 0.6-1.2 mL / min (0.8 mL / min); and / or

[0015] Injection volume: 5-15 μL (e.g. 10 μL).

[0016] In one embodiment, the liquid chromatography is a high performance liquid chromatography, and setting the detection wavelength on the high performance liquid chromatography comprises:

[0017] Diaminopyrimidine oxide: 287nm;

[0018] Pyrrolidinodiaminopyrimidine oxide: 228nm;

[0019] Metformin: 233 nm; and

[0020] Cloprostenol isopropyl ester: 228nm.

[0021] In one embodiment, the test sample comprises a pretreatment step before detection: ultrasonic treatment with 90% methanol, then adding n-hexane to mix, standing for stratification and taking the clear liquid as the test sample solution; preferably, the test sample solution is filtered through a filter membrane.

[0022] In one embodiment, the sample to be tested is selected from at least one of aqueous solutions, oils, creams, emulsions, gels and powders, and the sample to be tested includes a pretreatment step before testing: using 90% methanol to ultrasonically treat the sample to be tested, then adding n-hexane to mix, standing and stratifying, and taking the clear liquid as the sample solution to be tested; preferably, the sample solution to be tested is filtered through a filter membrane.

[0023] In one embodiment, the sample to be tested is selected from at least one of aqueous solutions, oils, creams, emulsions, gels and powders, and the sample to be tested includes a pretreatment step before detection: weigh 1g of the sample to be tested and place it in a stoppered colorimetric tube, accurately add 20ml of 90% methanol solution, vortex mix, ultrasonic treatment (preferably the ultrasonic treatment time is not less than 10-20min (for example, 15min)), add 10ml of n-hexane, vortex oscillation (preferably the vortex oscillation time is not less than 1min), centrifuge at 10000rpm for 5min, then stand and separate, take the clear liquid and filter it through a 0.45μm filter membrane to obtain the sample solution to be tested.

[0024] In one embodiment, the sample to be tested is wax-based, and the sample to be tested includes a pretreatment step before testing: using tetrahydrofuran to treat the sample to be tested, then adding 90% methanol solution to ultrasonically treat the sample to be tested, then adding n-hexane to mix, and after standing and stratifying, taking the clear liquid as the sample to be tested solution; preferably, the sample to be tested solution is filtered through a filter membrane.

[0025] In one embodiment, the sample to be tested is wax-based, and the sample to be tested includes a pretreatment step before detection: weigh 1g of the sample to be tested and place it in a stoppered colorimetric tube, accurately add 1ml of tetrahydrofuran solution, vortex mix to dissolve the sample, then accurately add 19ml of 90% methanol solution, vortex mix, ultrasonic treatment (preferably the ultrasonic treatment time is not less than 10-20min (for example, 15min)), add 10ml of n-hexane, vortex oscillation (preferably the vortex oscillation time is not less than 1min), centrifuge at 10000rpm for 5min, then stand and separate, take the lower clear liquid and filter it through a 0.45μm filter membrane to obtain the sample solution to be tested.

[0026] In one embodiment, the method comprises the steps of:

[0027] Step 1, using methanol as a solvent to prepare standard solutions of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester with different concentrations;

[0028] Step 2, using high performance liquid chromatography to detect the different concentrations of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester standard solutions obtained in step 1, and drawing standard curves of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester according to the concentrations and instrument response values ​​of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester;

[0029] Step 3, using the same test conditions as step 2 to detect the sample solution to be tested, and calculating the contents of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester respectively according to the standard curve obtained in step 2;

[0030] The chromatographic conditions of step 2 include: using an aqueous solution of phosphoric acid as the aqueous phase and methanol as the organic phase for gradient elution, and the procedure of the gradient elution is as follows:

[0031] Time (min) Water phase (v / v%) Organic phase (v / v%) 0 90 10 20 10 90 30 10 90 30.01 90 10 35 90 10 .

[0032] In one embodiment, the concentrations of the different concentrations of the standard solutions of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester are selected from at least three of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL, more preferably at least four, and more preferably at least five.

[0033] In one embodiment, the cosmetic is an aqueous solution, an oil, a cream, an emulsion, a wax-based solution, a gel or a powder.

[0034] In one embodiment, the cosmetic is a hair cleansing or care product, such as a hair cleansing or care product, a beard cleansing or care product, an eyelash or eyebrow cleansing or care product.

[0035] In one embodiment, the cosmetics are shampoo, shower gel, conditioner, hair dye, perm cream, hair mask, hair care essential oil, hair wax, styling spray, hair gel, hair cream, hair sunscreen spray, hair growth liquid, beard cream, beard oil, beard growth liquid, beard lotion, eyebrow dye, hair dye, eyebrow pencil, eyebrow powder, eyebrow growth liquid, eyelash growth liquid, eyelash styling liquid.

[0036] Based on the prior art, the present invention has at least the following advantages:

[0037] 1. The method of the present invention adopts targeted and personalized pre-treatment methods for different types of cosmetic matrixes. It can complete the detection of multiple compounds in a short time, reduce the sample pre-treatment steps, provide high-precision quantitative analysis, good data repeatability, high recovery rate, accurate test results, high credibility of results, and fill the gap in the determination of effective ingredients and prohibited components that promote hair growth in cosmetics.

[0038] 2. In the embodiment of the present invention, a variety of extraction solutions were screened, and 10% methanol solution was used to extract four compounds such as diaminopyrimidine oxide in the sample, and it was found that the sample recovery rate of cloprostenol isopropyl ester in the sample was basically 0%. Subsequently, 30% methanol, 50% methanol, 70% methanol, and 90% methanol solutions were used as extraction solutions in turn, and it was found that the sample recovery rate of 90% methanol solution was the highest, and 90% methanol solution was finally selected as the extraction solution.

[0039] 3. In the embodiment of the present invention, the conditions of the mobile phase were screened: methanol was used as the organic phase, and the separation effects of three different aqueous phase systems, 0.1% phosphoric acid, 1.7% diammonium phosphate (PH=3) and water, on the four compounds were investigated respectively. It was found that when the aqueous phase was water, diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, and metformin could all stably elute, but cloprostenol isopropyl ester was strongly retained on the chromatographic column and could not be eluted. When 1.7% diammonium phosphate (PH=3) was used as the aqueous phase, it was found that cloprostenol isopropyl ester could elute when the organic phase ratio was large, but the response value of the peak area was small and could not meet the sensitivity requirements of the determination. When we used 0.1% phosphoric acid as the aqueous phase, it was found that the four compounds could all elute, and the separation effects were very satisfactory, so 0.1% phosphoric acid and methanol were used as the mobile phase system.

[0040] 4. In the embodiments of the present invention, different pre-treatment methods are developed for different matrices: for wax-based, we found in our experiments that the recovery rate of some wax-based products using method 1 can reach more than 80%, but the sample recovery rate of most wax-based products is relatively low, only between 50-60%. Later, a dissolution step with tetrahydrofuran was added during the pre-treatment process, and it was found that the recovery rate was generally increased to 80-90%. Therefore, the present invention selects different pre-treatment methods for wax-based.

[0041] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0043] Figure 1 This is a high performance liquid chromatogram of a standard solution of diaminopyrimidine oxide (1) according to an embodiment of the present invention, with a detection wavelength of 287 nm;

[0044] Figure 2 The HPLC chromatogram of the standard solution of pyrrolidino diaminopyrimidine oxide (2) and cloprostenol isopropyl ester (4) according to the embodiment of the present invention, the detection wavelength is 228 nm;

[0045] Figure 3 The HPLC chromatogram of the metformin (3) standard solution according to the embodiment of the present invention, the detection wavelength is 233 nm;

[0046] Figure 4 is a high performance liquid chromatography-tandem mass spectrometry diagram of a standard solution of diaminopyrimidine oxide (1) according to an embodiment of the present invention;

[0047] Figure 5 is a high performance liquid chromatography-tandem mass spectrometry diagram of a standard solution of pyrrolidino diamino pyrimidine oxide (2) according to an embodiment of the present invention;

[0048] Figure 6 is a high performance liquid chromatography-tandem mass spectrometry diagram of a standard solution of metformin (3) according to an embodiment of the present invention;

[0049] Figure 7 is a high performance liquid chromatography-tandem mass spectrometry diagram of a standard solution of cloprostenol isopropyl ester (4) according to an embodiment of the present invention; DETAILED DESCRIPTION

[0050] After extensive and in-depth research, the inventors have developed a method for simultaneously detecting four compounds in cosmetics, namely, diaminopyrimidine oxide, pyrrolidinopyrimidine oxide, metformin and cloprostenol isopropyl ester. The method avoids derivatization by optimizing the pretreatment method, and optimizes the chromatographic conditions to achieve good recovery rate, linear range, accuracy and repeatability of the method, thereby making up for the defect that hair products lack a one-step detection method for prohibited substances such as diaminopyrimidine oxide, pyrrolidinopyrimidine oxide, metformin and cloprostenol isopropyl ester. The method can provide important technical support for the industry supervision of cosmetics and meet the needs of cosmetics quality and safety supervision.

[0051] To make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the present application is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually based on normal conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0052] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0053] Example 1

[0054] This example provides a method for detecting four compounds such as diaminopyrimidine oxide in cosmetics, and the method parameters are as follows:

[0055] 1. Reagents and Materials

[0056] Unless otherwise specified, all reagents used in this method are of analytical grade or above, and the water is the first-grade water specified in GB / T 6682.

[0057] 1.1. Standard products, see Table 1-1.

[0058] Table 1-1 Standard product information table

[0059]

[0060] Note: If the standard product is different from the target component, necessary conversion must be performed.

[0061] 1.2. Methanol, chromatographic grade.

[0062] 1.3. Tetrahydrofuran, analytical grade.

[0063] 1.4. Hexane

[0064] 1.5. Phosphoric acid (85%), extra pure.

[0065] 1.6. Formic acid, chromatographic grade.

[0066] 1.7.90% methanol: Accurately measure 900.0 mL of methanol (1.2), add ultrapure water to 1000 mL, mix well, and the mixture is ready.

[0067] 1.8.0.1% phosphoric acid solution: Accurately measure 1.0 mL of phosphoric acid (1.5), add ultrapure water to 1000 mL, mix well, and filter through a 0.45 μm filter membrane to obtain the solution.

[0068] 1.9.0.01% formic acid solution: Accurately measure 0.1 mL of formic acid (1.6), add ultrapure water to 1000 mL, mix well, and filter through a 0.45 μm filter membrane to obtain the solution.

[0069] 1.10. Standard stock mixed solution (ρ = 1 g / L): Accurately weigh 0.01 g (accurate to 0.0001 g) of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester quasi-substances (1.1) respectively into a 10 mL volumetric flask, dissolve with methanol (1.2) and dilute to the mark, shake well, and use as the standard stock mixed solution of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester. Store in a -18°C refrigerator away from light.

[0070] 1.11. Standard series solutions

[0071] Accurately pipette an appropriate amount of 1.10 standard stock mixed solution and dilute it with 90% methanol (1.7) to prepare a series of standard solutions with mass concentrations of 0.1, 0.5, 1, 5, 10, 50, 100 and 200 μg / mL for diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester.

[0072] 2. Instruments and Equipment

[0073] 2.1. High performance liquid chromatograph, UV detector.

[0074] 2.2. Analytical balance: sensitivity 0.0001g and 0.00001g.

[0075] 2.3. High-speed centrifuge (speed ≥ 10000rpm).

[0076] 2.4. Vortex mixer.

[0077] 2.5. Ultrasonic cleaner

[0078] 3. Sample Processing

[0079] Aqueous solutions, oils, creams, emulsions, gels or powders: Weigh 1 g (accurate to 0.001 g) of sample into a 50 mL stoppered colorimetric tube, accurately add 20 ml of 90% methanol (1.7), vortex mix, ultrasonically treat for 15 min, add 10 ml of n-hexane (1.4), vortex for 1 min, centrifuge at 10,000 rpm for 5 min, and filter the lower clear liquid through a 0.45 μm filter membrane as the test solution.

[0080] Wax-based: weigh 1 g of the sample to be tested and place it in a stoppered colorimetric tube, accurately add 1 ml of tetrahydrofuran (1.3), vortex mix to dissolve the sample, accurately add 19 ml of 90% methanol (1.7), vortex mix, ultrasonically treat (preferably the ultrasonic treatment time is not less than 10-20 min (e.g. 15 min)), add 10 ml of n-hexane (1.4), vortex oscillate (preferably the vortex oscillation time is not less than 1 min), centrifuge at 10000 rpm for 5 min, let stand to separate layers, take the lower supernatant and filter it through a 0.45 μm filter membrane to obtain the sample solution to be tested.

[0081] 4. Detection

[0082] The sample solution containing four compounds including diaminopyrimidine oxide and the standard series solution were connected to a high performance liquid chromatography system for detection.

[0083] HPLC conditions:

[0084] Chromatographic column: AQ-C18 column (((250mm×4.6mm×5μm), or equivalent chromatographic column;

[0085] Column temperature: 30°C;

[0086] Mobile phase: Solution A: 0.1% phosphoric acid solution (1.6); Solution B: methanol (1.2). Gradient elution program is shown in Table 1-2.

[0087] Table 1-2 Gradient elution program

[0088] Time (min) A(％) B(％) 0 90 10 20 10 90 30 10 90 30.01 90 10 35 90 10

[0089] Flow rate: 0.8 mL / min;

[0090] Injection volume: 10 μL;

[0091] Detection wavelength: diaminopyrimidine oxide: 287nm; pyrrolidine diaminopyrimidine oxide: 228nm; metformin: 233nm; cloprostenol isopropyl ester: 228nm.

[0092] The same HPLC conditions were used to detect diaminopyrimidine oxidation; pyrrolidinopyrimidine oxidation; metformin; cloprostenol isopropyl ester standards on a LC-MS instrument. The spectra are shown in the attached Figure 4-7 .

[0093] Example 2

[0094] In this embodiment, the sample spike recovery rate of the method is investigated. The positive sample spike determination method is to select six positive samples containing diaminopyrimidine oxide or pyrrolidine diaminopyrimidine oxide, perform positive spike tests on them, add the components to be tested in an amount equivalent to the sample, and determine the recovery rate results. The sample information is detailed in Table 2-1.

[0095] Table 2-1 Sample information

[0096] Sample name Label identification ingredients Matrix type Cosmetics A diaminopyrimidine oxide Liquid water Cosmetics B diaminopyrimidine oxide Oil Cosmetics C diaminopyrimidine oxide Creams and lotions Cosmetics D Pyrrolidino diaminopyrimidine oxide Liquid water Cosmetics Pyrrolidino diaminopyrimidine oxide Oil Cosmetics Pyrrolidino diaminopyrimidine oxide Creams and lotions

[0097] 1. Instruments and Reagents

[0098] Agilent 1260 high performance liquid chromatograph (Agilent, USA); Sartorius CP224S and 225D-1CN electronic balances (Sartorius, Germany); 5800 ultrasonic analyzer (Branson, USA); MS3 vortex mixer (IKA, Germany); 5810R desktop centrifuge (Eppendof, Germany); Milli-Q Reference A+ ultrapure water analyzer (Millipore, USA).

[0099] Diaminopyrimidine oxide standard (purity 95%, Bidex Pharmaceutical Co., Ltd.); pyrrolidine diaminopyrimidine oxide (purity 97%, Bidex Pharmaceutical Co., Ltd.); metformin (purity 100%, China Food and Drug Administration); cloprostenol isopropyl ester standard (purity 98%, Shanghai MacLean Biotechnology Co., Ltd.); methanol (chromatographic grade, Merk, Germany); phosphoric acid (analytical grade, Shanghai Lingfeng Chemical Reagent Co., Ltd.); tetrahydrofuran (analytical grade, Fisher, USA); n-hexane (analytical grade, Shanghai Lingfeng Chemical Reagent Co., Ltd.); formic acid (chromatographic grade, Shanghai Anpu Experimental Technology Co., Ltd.); Shanghai Titan Technology Co., Ltd. All water was ultrapure water.

[0100] Chromatographic conditions

[0101] Chromatographic column: AQ-C18 column (((250mm×4.6mm×5μm), or equivalent chromatographic column;

[0102] Column temperature: 30°C;

[0103] Mobile phase: Solution A: 0.1% phosphoric acid solution (1.8); Solution B: methanol (1.2). Gradient elution program is shown in Table 2-2.

[0104] Table 2-2

[0105] Time (min) A(％) B(％) 0 90 10 20 10 90 30 10 90 30.01 90 10 35 90 10

[0106] Flow rate: 0.8 mL / min;

[0107] Injection volume: 10 μL;

[0108] Detection wavelength: diaminopyrimidine oxide: 287nm; pyrrolidine diaminopyrimidine oxide: 228nm; metformin: 233nm; cloprostenol isopropyl ester: 228nm.

[0109] 3. Determination of linear relationship in HPLC system:

[0110] Standard stock solutions of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin, and cloprostenol isopropyl ester (1 mg / mL): Accurately weigh 10 mg of each standard substance of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin, and cloprostenol isopropyl ester, respectively, in a 10 mL volumetric flask, dissolve with methanol and dilute to the mark, shake well, and prepare standard stock solutions of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin, and cloprostenol isopropyl ester with a concentration of 1 mg / mL. Store in a -20°C refrigerator away from light.

[0111] Standard series solutions: Accurately pipette an appropriate amount of standard stock solution and dilute it with 90% methanol to prepare a series of standard solutions with mass concentrations of 0.1, 0.5, 1, 5, 10, 50, 100 and 200 μg / mL for diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester.

[0112] Linear regression analysis was performed with the peak area as the ordinate (y) and the concentration as the abscissa (x, μg / mL), and the linear equation was obtained as shown in Table 2-3 below.

[0113] Table 2-3

[0114]

[0115] 4. Treatment of sample solution for spiked recovery of positive samples: Take 1.0g (accurate to 0.001g) of diaminopyrimidine oxide positive sample and pyrrolidinyl diaminopyrimidine oxide positive sample respectively, accurately add diaminopyrimidine oxide or pyrrolidinyl diaminopyrimidine oxide standard equivalent to the amount in the sample, place in a 50mL stoppered colorimetric tube, accurately add 20ml of 90% methanol solution, vortex mix, ultrasonic treatment for 15min, add 10ml of n-hexane solution, vortex oscillation for 1min, centrifuge at 10000rpm for 5min, filter the lower clear liquid through a 0.45μm filter membrane, and use it as the test solution. Prepare the test solution according to the law as the recovery solution, repeat the experiment 6 times for each spike level, and calculate the recovery and relative standard deviation (n=6).

[0116] The content of diaminopyrimidine oxide and pyrrolidine diaminopyrimidine oxide in the sample is calculated according to formula (1):

[0117] (1)

[0118] Where: ω——the content of diaminopyrimidine oxide and pyrrolidine diaminopyrimidine oxide in cosmetics, %;

[0119] m——sample sampling volume, g;

[0120] ρ——the mass concentration of diaminopyrimidine oxide and pyrrolidine diaminopyrimidine oxide in cosmetics in the sample calculated by substituting into the standard curve, μg / mL;

[0121] V——fixed volume, mL.

[0122] Under chromatographic conditions, the standard working curve solution and the sample solution were sampled separately, and qualitative analysis was performed based on the retention time and the spectrum of the diode array. The content of diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide in the sample solution was obtained from the standard curve. The response values ​​of diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide in the sample solution should be within the linear range of the standard curve. If the linear range is exceeded, the extract should be diluted and measured or the amount of the extract should be increased and retested. The results of the positive spike recovery rate determination are shown in Tables 2-4 and 2-5, and the results show that the recovery rate determination obtained satisfactory results.

[0123] Table 2-4 Results of positive spike recovery tests of diaminopyrimidine oxides in different matrices

[0124]

[0125] Table 2-5 Results of positive spike recovery tests on different matrices of pyrrolidine diaminopyrimidine oxide

[0126]

[0127]

[0128] Example 3

[0129] Blank sample spike determination: Take six blank matrix samples of liquid aqueous preparations, creams and lotions, wax-based samples, oily samples, gels, and powders that do not contain the four compounds such as diaminopyrimidine oxide, and perform blank spike tests on them to determine the recovery results.

[0130] 1. Instruments and Reagents

[0131] Same as above-mentioned embodiment 1.

[0132] 2. Chromatographic conditions

[0133] Same as above-mentioned embodiment 1.

[0134] 3. Determination of linear relationship in HPLC system

[0135] Same as above-mentioned embodiment 1.

[0136] 4. Blank sample spike recovery sample processing:

[0137] Water, oil, cream, emulsion, gel or powder: weigh 1g of sample (accurate to 0.001g) and place it in a 50mL stoppered colorimetric tube, accurately add 20ml of 90% methanol, vortex mix, ultrasonic treatment for 15min, add 10ml of n-hexane, vortex oscillation for 1min, centrifuge at 10000rpm for 5min, filter the lower clear liquid through a 0.45μm filter membrane, and use it as the test solution. Prepare the test solution according to the law as the recovery solution, repeat the experiment 6 times for each spike level, and calculate the recovery and relative standard deviation (n=6).

[0138] Wax-based: Weigh 1g of the sample to be tested and place it in a stoppered colorimetric tube, accurately add 1ml of tetrahydrofuran, vortex mix to dissolve the sample, then accurately add 19ml of 90% methanol, vortex mix, ultrasonically treat for 15min, add 10ml of n-hexane, vortex oscillate for 1min, centrifuge at 10000rpm for 5min, and filter the lower clear liquid through a 0.45μm filter membrane as the test solution. Prepare the test solution according to the law as the recovery solution, repeat the experiment 6 times for each spike level, and calculate the recovery and relative standard deviation (n=6).

[0139] The calculation of four compounds including diaminopyrimidine oxide in the sample is calculated according to formula (1):

[0140] (1)

[0141] Where: ω——Contents of four compounds including diaminopyrimidine oxide in cosmetics, %;

[0142] m——sample sampling volume, g;

[0143] ρ——The mass concentration of four compounds including diaminopyrimidine oxide in cosmetics in the sample calculated by substituting into the standard curve, μg / mL;

[0144] V——fixed volume, mL.

[0145] Under chromatographic conditions, the standard working curve solution and the sample solution are sampled separately, and qualitative analysis is performed based on the retention time and the spectrum of the diode array. The contents of four compounds such as diaminopyrimidine oxide in the sample solution are obtained from the standard curve. The response values ​​of the four compounds such as diaminopyrimidine oxide in the sample solution should be within the linear range of the standard curve. If the response values ​​exceed the linear range, the extract solution should be diluted and then measured or the amount of the extract solution should be increased and re-tested.

[0146] The results of the positive spike recovery rate determination are shown in Table 3-1, and the results show that the recovery rate determination obtained satisfactory results.

[0147] Table 3-1 Recovery and precision test results of blank matrix spiked

[0148]

[0149]

[0150] Example 4

[0151] 1. Instruments and Reagents

[0152] Same as above-mentioned embodiment 1.

[0153] 2. Chromatographic conditions

[0154] Same as above-mentioned embodiment 1.

[0155] 3. Determination of linear relationship in HPLC system

[0156] Same as above-mentioned embodiment 1.

[0157] 4. Sample treatment: aqueous solutions, oils, creams, emulsions, gels or powders: weigh 1g of the sample to be tested and place it in a stoppered colorimetric tube, accurately add 20ml of 90% methanol solution, vortex mix, ultrasonically treat for 15min, centrifuge at 10000rpm for 5min, and filter through a 0.45μm filter membrane to obtain the sample solution to be tested.

[0158] The calculation of four compounds including diaminopyrimidine oxide in the sample is calculated according to formula (1):

[0159] (1)

[0160] Where: ω——Contents of four compounds including diaminopyrimidine oxide in cosmetics, %;

[0161] m——sample sampling volume, g;

[0162] ρ——The mass concentration of four compounds including diaminopyrimidine oxide in cosmetics in the sample calculated by substituting into the standard curve, μg / mL;

[0163] V——fixed volume, mL.

[0164] Under chromatographic conditions, the standard working curve solution and the sample solution are sampled separately, and qualitative analysis is performed based on the retention time and the spectrum of the diode array. The contents of four compounds such as diaminopyrimidine oxide in the sample solution are obtained from the standard curve. The response values ​​of the four compounds such as diaminopyrimidine oxide in the sample solution should be within the linear range of the standard curve. If the response values ​​exceed the linear range, the extract solution should be diluted and then measured or the amount of the extract solution should be increased and re-tested.

[0165] The sample determination results are shown in Table 4-1. The extraction method and rapid determination method described in the present invention can accurately quantify the contents of four compounds such as diaminopyrimidine oxide in the sample.

[0166] Table 4-1 Determination results of four compounds including diaminopyrimidine oxide in samples

[0167] Cosmetic Name Label Ingredients Measured content (%) Cosmetics 1 diaminopyrimidine oxide 1.025 Cosmetics 2 Pyrrolidino diaminopyrimidine oxide 0.895

[0168] In summary, the detection method of the four compounds such as diaminopyrimidine oxide in cosmetics of the present invention is simple and rapid to operate, with high sensitivity, and its recovery rate and repeatability meet the requirements of daily detection. The method has high sensitivity, high mass accuracy and feasible linear range, which greatly improves the monitoring ability of the detection agency for cosmetics.

[0169] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present application, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present application.

Claims

1. A method for simultaneously detecting diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester in cosmetics, characterized in that: The method comprises the steps of: using liquid chromatography to detect whether the cosmetic to be tested contains diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester; wherein the chromatographic conditions of the liquid chromatography comprise: using an aqueous solution of phosphoric acid as an aqueous phase and using methanol as an organic phase for gradient elution, and the procedure of the gradient elution is as follows: 。 2. The method according to claim 1, characterized in that The mass concentration of phosphoric acid in the aqueous solution of phosphoric acid is 85%.

3. The method according to claim 1, characterized in that The chromatographic conditions also include: Chromatographic column: C18 column (preferably AQ-C18 column); Column temperature: 25-35°C (e.g. 30°C); Flow rate: 0.6-1.2 mL / min (0.8 mL / min); and / or Injection volume: 5-15 μL (e.g. 10 μL).

4. The method according to claim 1, characterized in that: The liquid chromatography is a high performance liquid chromatography, and setting the detection wavelength on the high performance liquid chromatography includes: Diaminopyrimidine oxide: 287nm; Pyrrolidinodiaminopyrimidine oxide: 228nm; Metformin: 233 nm; and Cloprostenol isopropyl ester: 228nm.

5. The method according to claim 1, characterized in that The sample to be tested is selected from at least one of aqueous solutions, oils, creams, emulsions, gels and powders. The sample to be tested includes a pretreatment step before testing: using 90% methanol to ultrasonically treat the sample to be tested, then adding n-hexane to mix, standing and stratifying, and taking the clear liquid as the sample solution to be tested; preferably, the sample solution to be tested is filtered through a filter membrane.

6. The method according to claim 1, characterized in that The sample to be tested is wax-based, and the sample to be tested includes a pretreatment step before testing: using tetrahydrofuran to treat the sample to be tested, then adding 90% methanol solution to ultrasonically treat the sample to be tested, then adding n-hexane to mix, and after standing and stratifying, taking the clear liquid as the sample solution to be tested; preferably, the sample solution to be tested is filtered through a filter membrane.

7. A method for simultaneously detecting diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester in cosmetics, characterized in that: The method comprises the steps of: Step 1, using methanol as a solvent to prepare standard solutions of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester with different concentrations; Step 2, using high performance liquid chromatography to detect the different concentrations of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester standard solutions obtained in step 1, and drawing standard curves of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester according to the concentrations and instrument response values ​​of diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester; Step 3, using the same test conditions as step 2 to detect the sample solution to be tested, and calculating the contents of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester respectively according to the standard curve obtained in step 2; Wherein, the chromatographic conditions of the liquid chromatography are as described in any one of claims 1-6.

8. The method according to claim 7, characterized in that The concentrations of the different concentrations of the standard solutions of diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, metformin and cloprostenol isopropyl ester are selected from at least three of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL.

9. The method according to any one of claims 1 to 8, characterized in that The cosmetics are water-based, oil-based, cream-based, emulsion-based, wax-based, gel-based or powder-based.

10. The method according to claim 9, characterized in that The cosmetics include shampoo, shower gel, conditioner, hair dye, perm cream, hair mask, hair essential oil, hair wax, styling spray, hair gel, hair cream, hair sunscreen spray, hair growth liquid, beard cream, beard oil, beard growth liquid, beard lotion, eyebrow dye, hair dye, eyebrow pencil, eyebrow powder, eyebrow growth liquid, eyelash growth liquid, and eyelash styling liquid.

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