Determination of glutathione in skin care products by fluorescence spectrophotometry based on passivated carbon quantum dots
Abstract
A fluorescence spectrophotometric method for the determination of glutathione in skin care products based on passivated carbon quantum dots was established.
Carbon quantum dot solution was prepared by heating citric acid at high temperature. Glutathione standard solution with different concentrations was mixed with 1-ethyl – (3-dimethylaminopropyl) carbon diimide solution (EDC) for 10 min, and then added into the carbon quantum dot solution for 10 min.
The samples were extracted with water, diluted with PBS phosphate buffer solution, and then operated synchronously with standard solution to determine fluorescence intensity.
The mass concentration of glutathione solution was linear in the range of 0.1 ~ 100 μg/mL, the correlation coefficient was 0.999 4, and the detection limit was 0.004%.
The average recoveries were 93.7%-101.5% and the relative standard deviations were 1.3%-3.3% (n=6).
The method is simple and suitable for the rapid determination of glutathione in skin care products.
With the improvement of consumers’ attention to health and the increase of attention to skin care ingredients, the market for diversified, with clear efficacy and rapid response to the growing demand for skin care products, this trend has gradually become the core direction of skin care research and development.
Peptides are widely concerned functional components.
This class of molecules consists of protein fragments, formed through dehydration and polymerization processes, and connected to each other by chemical bonds (amide bonds).
They not only have the ability to regulate physiological state and nutritional function, but also play a crucial role in human growth and development cycle, nutrient metabolism, immune regulation mechanism and endocrine balance.
Glutathione is one of the first naturally active molecules to be applied to skin care products.
It is a tripeptide compound composed of three amino acids, glutamic acid, cysteine and glycine, and its structure contains special active groups, which can effectively remove free radicals in the body, so as to resist oxidation, improve skin color, fade color spots and slow down aging.
The main detection methods of glutathione include liquid chromatography, liquid chromatography-tandem mass spectrometry, capillary electrophoresis, electrochemical method and fluorescence method, among which chromatography, mass spectrometry and fluorescence method are widely used.
At present, the detection of glutathione in skin care products is mostly by chromatography and mass spectrometry, which has high sensitivity, but also requires high equipment.
Because of its advantages such as simple operation process, low instrument cost and fast reaction speed, fluorescence method has been widely concerned in scientific research and industrial fields.
However, the detection of glutathione by fluorescence method generally focuses on biological samples (such as serum, etc.), and its application in the detection of skin care products has not been reported.
The results show that the fluorescence intensity of carbon quantum dots obtained from citric acid heated at high temperature can be improved by passivating 1-ethyl – (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).
EDC molecules are introduced into the surface of carbon quantum dots, which promotes the trap emission of surface energy levels, and thus enhances the fluorescence properties of carbon quantum dots.
By pre-mixing glutathione with the EDC solution, glutathione consumes part of the EDC after binding with the EDC, and the remaining EDC continues to participate in the passivation of the carbon quantum dots, resulting in changes in the fluorescence intensity of the carbon quantum dots.
The results show that there is a linear relationship between the concentration of glutathione and the fluorescence intensity of carbon quantum dots after passivation, so as to realize the detection of glutathione.
Based on the characteristics of skin care products, a fluorescence spectrophotometric method for the determination of glutathione in skin care products was established for the first time.
The method is simple to operate, low cost of testing, and provides technical reference for raw material control and protection of consumer rights and interests.
Experimental part
1. Main instruments and reagents
- Fluorescence spectrophotometer: RF-6000 type.
- Electronic analytical balance: BSA224S type, sensitivity is 0.1mg.
- VORTEX oscillator: Kylin-Bell Vortex 5.
- High temperature furnace: BF51866C-1 type.
- pH meter: PHS-3C type.
- High speed centrifuge: 3-18KS type.
- Magnetic stirrer: MS7-H550-Pro type.
- Citric acid: Analytically pure.
- 1-ethyl – (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) : Purity (mass fraction) is 98%.
- PBS phosphate buffer solution: 0.01 mol/L, pH=7.0.
- Sodium hydroxide: Analytically pure.
- EDC solution: Weigh an appropriate amount of EDC and dissolve it with PBS phosphate buffer solution (pH=7.0) to prepare an EDC solution with a mass concentration of 50 mg/mL.
- Glutathione (reduced) standard material: purity (mass fraction) is 99.9%.
- The experimental water was grade I water.
- Skin care samples: commercially available.
2. Experimental procedure
(1) Preparation of carbon quantum dot solution
Weigh 2.00g (accurate to 0.01g) citric acid into 15 mL small beaker with lid and heat the small beaker in a high temperature furnace at 180 ℃.
After 40 minutes, the citric acid from a solid into a light yellow liquid, the small beaker from the high temperature furnace, cool to room temperature, at this time the small beaker in the light yellow liquid solidified.
Add 2 mL of 100 mg/mL sodium hydroxide solution into a small beaker, dissolve it completely under magnetic stirring, and then transfer it to a 10 mL volumetric bottle, fill it with 100 mg/mL sodium hydroxide solution, shake well, and then obtain a carbon quantum dot solution with a pH value of 7 for use.
(2) Solution preparation
Weigh 50.0 mg of glutathione reference material in a small beakers, add PBS phosphate buffer solution (pH=7.0), dissolve it, transfer it to a 50 mL volumetric bottle, mix it well, and prepare a standard reserve solution with a mass concentration of 1.0 mg/mL.
The appropriate volume of standard reserve solution was accurately removed to a 10 mL volumetric bottle, diluted with PBS phosphate buffer solution (pH=7.0), and the volume was fixed to prepare a series of standard working solutions with mass concentrations of 0.1, 0.5, 1.0, 20.0, 40.0, 60.0, 80.0, 100.0 μg/mL, respectively.
(3) Sample pretreatment
Weigh the skin care product sample 1.0g (accurate to 0.001g) into a 10 mL stopper colouring tube,
add 4 mL water and 1 g sodium chloride, swirl and oscillate on a vortex oscillator for 30 s, fix the volume with water to the line, and shake well.
Centrifuge for 5 min at 4 000 r/min.
1.0 mL supernatant accurately removed to 10 mL colorimetric tube with stopper, and the sample solution prepared by shaking with PBS phosphate buffer solution (pH=7.0) to 10 mL.
(4) Pre-processing of on-machine detection
Accurately remove 5.0 mL PBS phosphate buffer solution (pH=7.0) (corresponding to F0), 5.0 mL standard working solution with different concentrations (corresponding to F) and 5.0 mL sample solution,
add 50 μL EDC solution (50 mg/mL) each, and swirl and mix on the vortex oscillator for 10 min.
Add 30 μL carbon quantum dot solution and react for 10 min, to measured.
(5) Instrument operating condition
The wavelength of excitation light is 360 nm, the slit width of excitation and emission is 10 nm,
the scanning speed is 2 000 nm/min, and the emission spectrum is scanned and recorded in the range of 380 ~ 600 nm.
(6) Establishment of standard curve
Under the working conditions of the 1.2.5 instrument, the fluorescence intensity of each solution in 1.2.4 at the emission wavelength of 460 nm recorded.
The fluorescence intensity of the system without adding the standard solution was F0, and that of the system after adding the standard working solution was F.
The standard working curve drawn with the mass concentration of glutathione as the abscissa and F/F0 as the ordinate.
Results and discussion
Choice of reaction time
Accurately remove 5.0 mL PBS phosphate buffer solution (pH=7.0), add 50 μL of 50 mg/mL EDC solution to mix, and then add 30 μL carbon quantum dot solution to record fluorescence intensity at different reaction times, as shown in Figure 1.
As can seen from Figure 1, the fluorescence intensity increased sharply within the first 10 minutes.
After 10 min of reaction, the fluorescence intensity of the system basically stable,
indicating that the reaction between EDC and carbon quantum dots basically completed, so 10 min selected as the reaction time.
pH optimization
The effects of different pH values on the fluorescence intensity of the system investigated, and the results shown in Figure 2.
As can seen from Figure 2, when the pH value of the solution is less than 7, the fluorescence intensity is small.
When the pH value of solution is greater than 7, the fluorescence intensity is larger.
When pH is 7.0, the fluorescence intensity is the highest, indicating that EDC has the strongest passivation effect on carbon quantum dots at this time, so pH is 7.0 as the reaction environment of the experiment.
In order to reduce the influence of pH fluctuations on the experiment, a PBS phosphate buffer solution with pH 7.0 selected as the solvent for preparing glutathione and EDC, and a PBS phosphate buffer solution with pH 7.0 used as the diluent during sample pretreatment.
Coexisting test
To investigate the effects of common co-existing substances L-lysine, L-serine, L-carnosine, 1, 3-propanediol, 1, 2-propanediol, squalane, phenoxyethanol, methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate and butyl 4-hydroxybenzoate on the determination of glutathione.
The above substances prepared with PBS phosphate buffer solution (pH=7.0) with a mass concentration of 100 μg/mL.
Instead of glutathione standard solution, the operations were performed according to steps 1.2.4 and 1.2.5.
The F/F0 values under different coexisting substances were shown in Figure 3.
As can seen from Figure 3, F/F0 values of all other substances except glutathione are close to 1.0,
indicating that only glutathione can consume EDC, which indirectly affects the passivation degree of carbon quantum dots and makes carbon quantum dots show different fluorescence intensity,
indicating that this method has strong specificity for the determination of glutathione in skin care products.
1 – blank; 2-glutathione; 3-L-lysine; 4-L-serine; 5-L-carnosine; 6-1, 3-propanediol; 7-1, 2-propylene glycol; 8-squalane; 9 – phenoxyethanol; Methyl 10-4-hydroxybenzoate; Ethyl 11-4-hydroxybenzoate; 12-4-hydroxybenzoate propyl ester; Butyl 13-4-hydroxybenzoate Figure 3 F/F0 values for different coexisting substances
Linear equation, detection limit and quantitation limit
In 1.2.5 instrument operating conditions, follow step 1.2.6 to establish a standard operating curve.
The fluorescence spectra of the system after adding different concentrations of glutathione shown in Figure 4.
It can seen from Figure 4 that the fluorescence intensity of the system at the emission wavelength of 460 nm gradually decreases with the increase of the concentration of glutathione, and the position of the maximum emission peak the same, both located at 460 nm, and the fluorescence intensity F at 460 nm recorded.
The mass concentration of glutathione linearly correlated with F/F0 in the range of 0.1 ~ 100 μg/mL.
Linear regression performed using the mass concentration of glutathione as the abscess (x) and F/F0 as the abscess (y).
The linear equation was calculated as F/F0 = -0.0043x +0.969 8. The linear correlation coefficient was 0.999 4.
Where F is the fluorescence intensity of glutathione with different concentration and EDC solution (50 mg/mL) after the addition of carbon quantum dots, F0 is the fluorescence intensity of the system without glutathione.
The fluorescence intensity at 460 nm measured for 11 times without glutathione, and the detection limit 0.4 μg/mL calculated by the ratio of 3 times standard deviation to the slope of linear regression equation.
Since the method takes 1 g sample when testing the sample, after 1.2.3 pretreatment, it is equivalent to diluting the sample 100 times when testing on the machine, so the detection limit of the method is 0.004%, and the quantitative limit of the method is 0.012% when calculating the detection limit of the method by 3 times.
Standard recovery test
Three representative samples of water, cream and emulsion selected for recovery and precision tests.
Samples without glutathione weighed, and standard solutions with high and low levels added to the samples for 6 parallel tests respectively.
The relative standard deviations of recovery rates and measured values shown in Table 1.
As can seen from Table 1, the average recovery rate of glutathione ranged from 93.7% to 101.5% under the three concentrations, and the relative standard deviation of six parallel measurements ranged from 1.3% to 3.3%,
indicating that the method had high precision and accuracy, and could accurately determine glutathione content in skin care products.
Conclusion
A fluorescence spectrophotometric method for the determination of glutathione in skin care products based on passivated carbon quantum dots established.
The method has a wide linear range, good precision and accuracy, fast analysis speed and low detection cost, and can provide a strong reference for the detection of glutathione in skin care products, enhance the quality management and control of raw materials, and ensure that the legitimate rights and interests of consumers effectively protected.
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