2024-10-22
Determination of 6-methylcoumarin in Cosmetics by Liquid Chromatography
1.1 Instrument Configuration
Table 1 Configuration List of Liquid Chromatography
| No. | Module | Qty |
| 1 | PB3210 Binary Pump | 1 |
| 2 | CT3400 Column Oven | 1 |
| 3 | AS3210 Autosampler | 1 |
| 4 | UV3210 UV Detector | 1 |
| 5 | NovaChrom SC18 4.6*250mm, 5μm | 1 |
| 6 | SmartLab Workstation | 1 |
1.2 Experiment Method
1.2.1 Reagents
Table 2 List of Reagents
| No. | Reagents | Purity |
| 1 | Methanol | Chromatographic Grade |
| 2 | 6-methylcoumarin | 99% |
| 3 | Ammonium dihydrogen phosphate | AR |
| 4 | Phosphoric acid | GR |
1.2.1.1 6-Methylcoumarin standard stock solution (1000mg/L): Take appropriate amount of 6-methylcoumarin standard, dissolved and fix the volume with methanol and prepared into a concentration of 1000mg/L standard stock solution.
1.2.1.2 6-Methylcoumarin standard working solution: Pipette appropriate amount of 6-methylcoumarin standard stock solution and diluted with methanol to prepared a series of working curves with concentrations of 0.1mg/L, 0.5mg/L, 1.0mg/L, 3.0mg/L, 5.0mg/L and 10.0mg/L respectively.
1.2.1.3 Sodium dihydrogen phosphate buffer solution: Take 3.12g of sodium dihydrogen phosphate, add water to dissolve and dilute to 1000mL, and adjust the pH of phosphoric acid to 3.5.
1.2.2 Chromatography Conditions
Table 3 Chromatography Conditions
| Chromatography Column | NovaChrom SC18 4.6*250mm 5μm | ||
| Mobile Phase | A: Methanol，B：Sodium dihydrogen phosphate buffer solution | ||
| Flow Rate | 1mL/min | ||
| Column Temperature | 35°C | Wavelength | 275nm |
| Injection Volume | 10μL | ||
Table 4 Gradient Elution Program
| Time(min) | Mobile Phase A | Mobile Phase B |
| 0 | 55 | 45 |
| 11 | 55 | 45 |
| 12 | 90 | 10 |
| 40 | 90 | 10 |
| 41 | 55 | 45 |
| 50 | 55 | 45 |
1.2.3 Sample Pretreatment
Take 1g (accurate to 0.001g) of the sample in a 10mL volumetric flask, add 5mL of methanol, vortexing and shaking to fully mix the sample with the extraction solution, ultrasonic extraction for 20min, cooled to room temperature, and then fix the volume to 10mL with methanol, mixing and then transferring to centrifugal tubes, centrifuged at 5000r/min for 5min, and the supernatant filtered through the 0.45µm organic membrane, and then to be tested.
2. Experiment Result
2.1 System Suitability
Fig 1 10mg/L Chromatogram of Standards
Table 5 Test Data of 10mg/L Standards
| Compounds | Retention Time | Peak Area | Theoretical Plate Number |
| 6-methylcoumarin | 11.168 | 574.285 | 15854 |
Note：The chromatogram and data show that 6-methylcoumarin has a good peak shape, and there are no other peaks around the target peak, and the theoretical plate number is high, which meets the experimental requirements.
2.2 Standard Curve
Fig 2 Test Result of Standard Curve
Note: The chromatogram shows that the R value of the correlation coefficient of the 6-methylcoumarin curve is above 0.9999, which meets the experimental requirements.
2.3 Repeatability
Fig 3 Repeatability Chromatogram of 3mg/L Standards of 6 Injections
Table 6 Repeatability Data of 3mg/L Standards of 6 InjectionsTable 6 Repeatability Data of 3mg/L Standards of 6 Injections
|
6-methylcoumarin |
No. | Retention Time | Peak Area |
| 1 | 11.159 | 177.710 | |
| 2 | 11.161 | 176.711 | |
| 3 | 11.142 | 177.128 | |
| 4 | 11.152 | 176.985 | |
| 5 | 11.150 | 177.469 | |
| 6 | 11.149 | 177.629 | |
| RSD(%) | 0.061 | 0.222 |
Note: According to the data in the above table, the RSD of retention time repeatability of 6-methylcoumarin is 0.061%, and the RSD of peak area repeatability is 0.222%. The repeatability is good and meet the experimental requirements.
2.4 Detection Limit
Fig 4 Test Chromatogram of 0.02mg/L Standards
Table 7 Test Data of 0.02mg/L Standards
| Compounds | Retention Time | Peak Area | SNR |
| 6-methylcoumarin | 11.153 | 1.208 | 19.296 |
Note: According to the above data, 3 times signal-to-noise ratio is calculated as the detection limit, and it was found that the detection limit of 6-methylcoumarin is 0.004mg/L. It meets the experimental requirements.
2.5 Test Result of a Cosmetic Sample
Fig 5 Test Chromatogram of a Cosmetic Sample
Note: 6-Methylcoumarin was not detected in a cosmetic sample.
2.6 Attention
When using a high-speed centrifuge, take care that the tubes are symmetrically placed and the total mass of the tubes on opposite sides is the same.
3. Conclusion
The analytical method introduced in this article, with reference to the “Safety and Technical Standards for Cosmetics” in the detection of 6-methylcoumarin, by using Wayeal high performance liquid chromatography LC3200 series with UV detector. The experiment result shows that the peak shape of 6-methylcoumarin is good in the system adaptability test, and there are no other peaks around the target peak, and the theoretical plate number is high, which meets the experimental requirements. The curve correlation coefficient R value is above 0.9999. The RSD of retention time repeatability of 6-methylcoumarin is 0.061%, and the RSD of peak area repeatability is 0.222%, which shows good repeatability. The detection limit of 6-methylcoumarin is 0.004mg/L. All the above test results meet the requirements of the instrument in the standard method.
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