Abstract: Barbiturates are a class of barbituric acid derivatives mainly consisting of barbital, phenobarbital, amobarbital, and secobarbital. As central nervous system depressants, they possess sedative, hypnotic, and anticonvulsant effects. Overdose may lead to respiratory depression and even fatality. These compounds may also cause dizziness, drowsiness, and impaired fine motor coordination, and occasionally trigger severe allergic reactions such as exfoliative dermatitis. Long-term and continuous administration, particularly of phenobarbital, can induce hepatic drug-metabolizing enzymes and result in significant psychological and physical dependence. Accordingly, barbiturates are strictly prohibited in cosmetic products in China, the United States, the European Union, and many other regions. The prevalence of insomnia among Chinese adults now exceeds 50% and is gradually increasing in younger populations. Insomnia is closely associated with skin dryness and premature aging, which has boosted market demand for nourishing and sleep-improving cosmetics. To enhance their claimed efficacy, some unethical manufacturers illegally incorporate barbiturates into cosmetics, posing serious safety hazards. However, studies on the detection of barbiturates in cosmetics remain limited, and current standard methods often rely on matrix-matched calibration, which restricts their practical application. Therefore, it is highly desirable to develop a simple, rapid, and widely applicable analytical method for the routine monitoring of barbiturates in cosmetics. In this study, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established for the simultaneous determination of barbital, phenobarbital, amobarbital, and secobarbital in cosmetics. Samples were dispersed with n-hexane or ammonium acetate solution, followed by ultrasonic extraction with 80% methanol aqueous solution for 15 min. After centrifugation at 9 500 r/min, the upper n-hexane layer was discarded. An aliquot of 2.5 mL extract was mixed with 2.5 mL of 0.4% ammonia solution and purified by liquid-liquid extraction with 1 mL dichloromethane. Following vortex mixing for 2 min and high-speed centrifugation, the supernatant was filtered through a 0.22 μm organic filter prior to instrumental analysis. Chromatographic separation was performed on a Poroshell 120 EC C₁₈ column using gradient elution with water and methanol as the mobile phases. The flow rate was 0.3 mL/min, the column temperature was 30 °C, and the injection volume was 5 μL. Standard calibration curves were prepared using 40% methanol aqueous solution as the diluent. Detection was conducted in negative electrospray ionization mode under multiple reaction monitoring (MRM) mode. Quantification was performed using the external standard method based on peak area responses. All four barbiturates exhibited good linearity in their respective concentration ranges, with correlation coefficients (R²)≥0.995. The limits of detection (LODs) and quantification (LOQs) were 0.06 mg/kg and 0.20 mg/kg, respectively. At spiked levels of 0.20, 0.40, and 2.00 mg/kg, the average recoveries ranged from 80.7% to 108.1%, with relative standard deviations (RSDs, n=6) of 1.53%-9.79%. The validated method was applied to 10 commercially available cosmetic samples, and phenobarbital was detected in one sample at 0.615 mg/kg, confirming the potential risk of illegal barbiturate adulteration in cosmetics. The proposed method achieves satisfactory accuracy and precision with only two extraction steps. Compared with solid phase extraction and matrix solid phase dispersion, this approach is simpler, faster, and more cost-effective. In contrast to existing standard methods, it provides superior clean-up efficiency without requiring matrix-matched calibration and shows broader applicability. This reliable and practical method is well suited for the routine determination and regulatory monitoring of barbital, phenobarbital, amobarbital, and secobarbital in cosmetic products.

Key words: barbiturates, cosmetics, liquid-liquid extraction-high performance liquid chromatography-mass spectrometry (LLE-HPLC-MS/MS)