Simultaneous Determination of nicotinamide, kojic acid, Tranexamic acid, raspberry glycoside, azelaic acid, magnesium ascorbate phosphate and β-Arbutin in whitening cosmetics by UPLC-MS/MS

. A UPLC-MS/MS method was developed for simultaneous determination of 7 whitening ingredients: nicotinamide, kojic acid, Tranexamic acid, raspberry glycoside, azelaic acid, magnesium ascorbate phosphate and β-Arbutin in cosmetics. The whitening active components were extracted from cosmetics by supersonic extraction with sodium chloride and dichloromethane to disperse the sample, and supersonic extraction with 0.015 mol/L potassium dihydrogen phosphate solution purified by HLB solid phase extraction column, scanned and detected by electrospray ionization source with positive and negative ion alternate scanning mode and multiple reaction monitoring mode. The results showed that the whitening active ingredients were separated within 3 minutes, with a good linear relationship (R>0.999), and the detection limit was 0.10mg/kg~0.75mg/kg. The recoveries (n=6) were 78.84%-104.85%, and the RSDs were 0.24%-11.35%. This method is suitable for the rapid determination of whitening active ingredients in cosmetics.

Whitening and freckle-removing cosmetics have the effect of preventing spots and pigmentation caused by sunlight or other reasons, so they are favored by consumers. Its mechanism of action is to interfere or even block the formation of melanin. According to the principle of action of different chemicals, the active ingredients of whitening and freckle-removing can be classified into three categories [1]: one is as reducing agent, including vitamin C, E and derivatives; The second category is enzyme inhibitors: mainly inhibiting tyrosinase activity and affecting the transport of melanin, mainly represented by hydroquinone, azelaic acid, arbutin, kojic acid and derivatives; The third category is stripping agent: linoleic acid, citric acid, placenta extract, etc. Nicotinamide has been widely used in the medical and beauty fields with its anti-aging effect. It is believed that as a B-group vitamin compound, nicotinamide can achieve the effect of whitening skin by inhibiting the formation and transfer of melanin particles and accelerating the renewal of skin cells [2]. The cross and excessive use of these new whitening active ingredients will interfere with the normal enzyme conversion of tyrosine to melanin in the skin, cause adverse reactions to the skin, and even cause cancer [3].
At present, the commonly used methods for detecting whitening active ingredients in cosmetics include thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography-tandem mass spectrometry (GC-MS/MS), liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) [4][5], etc. Cosmetics are difficult to separate and purify due to the variety of raw materials and ingredients, complex matrix, and the removal and separation of impurities and interfering substances in the pretreatment will affect the accuracy of the results. Ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry has the advantages of high efficiency, fast, multicomponent analysis, high sensitivity, and accurate quantification. At present, there are few studies on the analysis and detection of whitening ingredients. In this study, three commonly used matrices in whitening cosmetics: aqueous solution, lotion, and face cream were investigated, and the chromatographic conditions for the separation of seven whitening active ingredients were optimized.

Preparation of standard solution
Accurately weigh NA, KA,TAA,RG, AA, MAP and AB standards 10mg (accurate to 0.1mg) is put into different 10mL brown volumetric flasks, and the chromatographypure methanol is dissolved to volume, and the concentration is 1000 μg/mL of single standard stock solution, stored under 4 ℃ in dark. Accurately transfer 0.05mL of single standard stock solution into the same 50mL volumetric flask, and obtain the concentration of 1 after the volume of methanol is fixed μ G/mL mixed standard intermediate solution, stored under 4 ℃ in dark. Take an appropriate amount of mixed standard intermediate solution, and prepare 0.1% oxalic acid solution into mixed standard working solution of appropriate concentration, which will be used on the machine and ready for use.

Extraction
Accurately weigh 1.000g of sample into a 50mL centrifuge tube with a stopper, add 2 g of sodium chloride and 5 mL of dichloromethane, place a ceramic homoproton, shake it with a vortex mixer until the sample is dispersed, and then add 20 mL of 0.015mol/L KH2PO4 solution for ultrasonic extraction for 30 minutes, and centrifuge at 8000 r/min for 3 minutes, transfer the upper supernatant into a 50mL volumetric flask, and extract the lower substances once with 20 mL of KH2PO4 solution, After the same centrifugation operation, combine the supernatant into the same volumetric flask, fix the volume to the scale, and wait for purification.

Purification
Transfer 4mL of the above extraction solution to the activated HLB column (200mg/6mL, activated successively with 3mL methanol and 3mL ultra-pure water in advance), control the flow rate to 1mL/min, collect the effluent, elute with 10mL 2% acetic acidmethanol solution, collect the eluate at 45 ℃, evaporate and concentrate it to near dry, dissolve the residue with 1.0mL 0.2% formic acid-acetonitrile solution, and pass 0.22 μ M filter membrane, UPLC-MS/MS analysis.

Optimize mass spectrum conditions
At the concentration of 1μg/mL, Seven kinds of mixed standard intermediate solution samples were scanned under different conditions of positive and negative ions, and the ionization mode was determined by the response strength of the ions. At the same time, the mass number of the precursor ion-product ion was optimized and screened successively in a triple four-stage bar to determine the quantitative and qualitative ion pairs, and the collision energy (CE) and retention time were optimized. Finally, the optimized mass spectrum conditions of the seven whitening active ingredients were obtained, The results are shown in Table 2. The typical MRM chromatogram detected under the optimal mass spectrum conditions is shown in Figure 1.

Linear relationship and detection limit
Use the mixed standard working solution series of different concentrations to inject samples under the optimal conditions to obtain the standard spectrum of whitening active ingredients of different concentrations. Take the concentration of each compound as the abscissa and the peak area of its corresponding quantitative ion pair as the ordinate to draw the standard curve, and obtain the linear range, regression equation and correlation coefficient R of seven whitening active ingredients, The detection limit LOD of 7 whitening active ingredients is calculated from the signal to noise ratio of their chromatograms greater than 3. Results All substances had a good linear relationship (R>0.999), and the detection limit was 0.10 mg/kg~0.75 mg/kg, as shown in Table 3.

Recovery and precision
Under the optimal conditions, select three kinds of negative samples, weigh appropriate amount of samples respectively, add mixed standard solution with three concentration gradients of high, middle and low, and carry out six parallel tests at each gradient level, and calculate the recovery rate and precision. The results showed that The recovery rates of nicotinamide, kojic acid, tranexamic acid, raspberry glycoside, azelaic acid, magnesium ascorbate phosphate and β-arbutin in water, lotion and face cream are 78.84%~104.85%, and the relative standard deviations (RSD) are 0.24%~11.35%, all less than 15%.

Contents of Whitening Ingredients in Commercial Products
Nine kinds of aqueous solutions, lotion and face cream with the words "whitening" on their labels purchased in major markets of Chongqing were determined. The results showed that among the nine samples, ascorbic acid phosphate salt was detected in No. 2 aqueous solution, but it was less than the detection limit, and could not be quantified; No. 5 lotion detected 20 mg/kg of tranexamic acid and 7.8 mg/kg of ursolic acid; Nicotinamide 25 mg/kg and azelaic acid 19 mg/kg were detected in No. 8 face cream.

Conclusion
In this paper, a method for simultaneous determination of seven whitening active ingredients in cosmetics by UPLC-MS/MS was established. The whole experiment is simple and fast, with high accuracy and sensitivity. It is suitable for the detection of multi-component whitening active ingredients in cosmetics with whitening and freckle removing effects, such as water, lotion, face cream, etc. It can achieve rapid qualitative and quantitative detection within 3 minutes, greatly shorten the detection time, improve the detection efficiency, and verify the linear equation The detection limit, recovery rate and precision indicators can meet the needs of daily market supervision, and provide technical support for preventing the risk of abuse of chemical substances and improving the supervision ability.