Bemegride (3-ethyl-3-methylglutarimide) is a central nervous system stimulant that directly excites the respiratory center and the vasomotor center, and antagonizes the effects of barbiturates and other hypnotics. However, this substance can cause severe adverse reactions such as visual and auditory hallucinations, and has been explicitly listed as a prohibited ingredient in cosmetics by the Safety and Technical Standards for Cosmetics (2015 Edition). To strengthen the quality and safety supervision of cosmetics, the State Administration for Market Regulation and the Standardization Administration of China issued GB/T 40898-2021 Determination of prohibited substances bemegride and its salts in cosmetics – High performance liquid chromatography, providing a unified technical basis for the determination of bemegride in cosmetics.
Based on the requirements of GB/T 40898-2021, this article systematically introduces the method principle, instrument configuration, chromatographic conditions, and validation results for the determination of bemegride and its salts in cosmetics using Elite’s HPLC solutions, providing technical support for cosmetic companies to establish a reliable quality control system.
Standard title: Determination of prohibited substances bemegride and its salts in cosmetics – High performance liquid chromatography
Scope: Applicable to the determination of bemegride and its salts in emulsion, cream, and lotion type cosmetics; results are expressed as bemegride.
Principle: After ultrasonic extraction, centrifugation, and filtration, the sample is analyzed by HPLC with a UV detector. Identification is based on retention time, and quantification is performed by the external standard method.
Sample preparation: For lotion type samples, directly weigh and extract with methanol; for cream/emulsion samples, add sodium chloride to break the emulsion before extraction.
Method detection and quantification limits: Results expressed as bemegride: detection limit 2 mg/kg, quantification limit 5 mg/kg.
GB/T 40898-2021 is a recommended national standard. However, once it is referenced by mandatory documents such as the Safety and Technical Standards for Cosmetics, the relevant parts become mandatory. Laboratories should obtain the official standard text and perform method verification before adopting this method.Based on GB/T 40898-2021, Elite has developed an HPLC solution suitable for the determination of bemegride and its salts in cosmetics.
I. Instrument Configuration
Elite EClassical 3200L HPLC system equipped with a UV detector.
II. Chromatographic Conditions
Column: Supersil ODS2 (5 μm, 4.6 × 150 mm)
Detection wavelength: 210 nm
Injection volume: 10 μL
Column temperature: 30 °C
Mobile phase: A – methanol; B – water (gradient program as below)
| Time (min) | A% (methanol) | B% (water) | Flow rate (mL/min) |
|---|---|---|---|
| 0 | 50 | 50 | 1.0 |
| 5.2 | 50 | 50 | 1.0 |
| 6.2 | 95 | 5 | 1.0 |
| 9.0 | 95 | 5 | 1.0 |
| 10.0 | 50 | 50 | 1.0 |
| 15.0 | 50 | 50 | 1.0 |
III. Experimental Method
1. Standard solution preparation
Bemegride standard stock solution (100 mg/L): Accurately weigh 10.0 mg of bemegride standard, dissolve and dilute to 100 mL with methanol. Store at 4 °C, valid for 3 months.
2. Standard working solutions
Dilute the stock solution stepwise with methanol to obtain 0.5, 1, 2, 5, and 10 mg/L.
3. Sample preparation (example for lotion type sample)
Weigh approximately 1 g of sample (accurate to 0.001 g) into a 10 mL stoppered colorimetric tube. Add methanol to volume, mix well, and sonicate for 20 min. Transfer to a centrifuge tube and centrifuge at 5000 r/min for 15 min. Pass the supernatant through a 0.45 μm membrane filter before analysis.
IV. Experimental Results
1. Typical chromatograms
Figures 1 and 2 show chromatograms of bemegride standard solutions (10 mg/L and 0.5 mg/L) under the above conditions.
Figure 1. Chromatogram of bemegride standard (10 mg/L)
Figure 2. Chromatogram of bemegride standard (0.5 mg/L)
2. Linearity and linear range
Following the method in GB/T 40898-2021, five standard solutions of bemegride at concentrations of 0.5, 1, 2, 5, and 10 mg/L were prepared. The linear regression equation was y = 38.8011x – 1.4981, with a correlation coefficient R² = 0.9997, indicating good linearity in the range of 0.5–10 mg/L.
3. Method repeatability
A 5 mg/L bemegride standard solution was injected five times consecutively. The RSD of retention time was 0.21%, and the RSD of peak area was 0.21%, both below the standard requirement of 1%, demonstrating good method repeatability.
4. Method detection and quantification limits
The baseline noise was 3.66 × 10⁻⁵ AU. The instrument detection limit (LOD) was calculated at S/N = 3, and the instrument quantification limit (LOQ) at S/N = 10. Taking into account the sample preparation dilution factor (1 g sample diluted to 10 mL, dilution factor ≈10), the method LOD and LOQ were calculated. The results are shown in Table 1. The method LOD of 0.31 mg/kg is below the standard requirement of 2 mg/kg, meeting detection needs.
Table 1. Method LOD (S/N=3) and LOQ (S/N=10)
| Parameter | Instrument LOD (mg/L) | Instrument LOQ (mg/L) | Method LOD (mg/kg) | Method LOQ (mg/kg) | GB/T 40898-2021 requirement for method LOD |
|---|---|---|---|---|---|
| Bemegride | 0.03 | 0.10 | 0.31 | 1.03 | 2 mg/kg |
5. Sample determination
Figure 3 shows chromatograms of a commercially available facial toner sample and a spiked facial toner sample (100 mg/kg bemegride). The results showed that bemegride was not detected in the original sample, and the spiked recoveries ranged from 90.7% to 105.7%, meeting the standard method requirements.
Figure 3. Chromatograms of facial toner samples for bemegride analysis
V. Conclusion
Based on GB/T 40898-2021, an HPLC method for the determination of bemegride in cosmetics was established using the Elite EClassical 3200L HPLC system and a Supersil ODS2 column. This solution fully meets the national standard requirements and can be used for the qualitative and quantitative analysis of bemegride and its salts in emulsion, cream, and lotion type cosmetics, providing reliable technical support for the quality and safety supervision of cosmetics.
