Estrogens, a class of sex hormones, play crucial roles in various physiological processes but their presence in cosmetics is prohibited due to potential adverse health effects, including metabolic dysfunctions, breast cancer, and endometrial hyperplasia. Existing detection methods, such as HPLC-UV and HPLC-MS, are often time-consuming and require complex sample pretreatment. Ambient ionization mass spectrometry (AIMS), particularly paper spray ionization mass spectrometry (PSI-MS), offers a rapid and less laborious alternative. However, the sensitivity of PSI-MS for certain compounds, including estrogens, can be limited. This study introduces reactive paper spray ionization mass spectrometry (RPSI-MS), combining the advantages of PSI-MS with online derivatization to improve detection sensitivity and simplify the analytical workflow for estrogen detection in cosmetics. The method employs FluMP as a derivatization reagent to react with estrogens, enhancing their ionization efficiency and improving detection sensitivity. This approach aims to provide a sensitive, rapid, and environmentally friendly method for the detection and quantification of estrogens in cosmetic products, addressing the limitations of existing techniques.

Several methods for detecting estrogens in cosmetics have been reported, including those based on excitation-emission matrix fluorescence and high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) or mass spectrometry (MS) detection. However, these methods often suffer from drawbacks such as time-consuming sample preparation procedures and lower sensitivity. Ambient ionization mass spectrometry (AIMS), such as paper spray ionization (PSI), has emerged as a promising technique for direct analysis of complex samples with minimal pretreatment. PSI-MS, combining the advantages of AIMS and electrospray ionization, has been applied to analyze various compounds. Recently, reactive paper spray ionization mass spectrometry (RPSI-MS) has been developed, integrating online derivatization to enhance the sensitivity of the detection of compounds with low mass spectrum responses. This technique has been shown to be effective for various compounds, including quinones and aldehydes. The current study builds upon these advancements, adapting RPSI-MS to the specific challenge of rapidly and sensitively detecting estrogens in cosmetic products.

The study employed RPSI-MS for the quantitative analysis of estradiol, estriol, and ethinylestradiol in cosmetics. FluMP was chosen as the derivatization reagent due to its reactivity with primary and secondary alcohols and phenols. Several parameters were optimized, including FluMP concentration and volume (5 µL of 1 mg/mL was found to be optimal), the addition of triethylamine (adding it to the estrogen solution at a v/v ratio of 1:10 provided the best results), and the drying time after sample application to the paper. Estradiol valerate served as the internal standard. Whatman grade 1 filter paper, cut into isosceles triangles, was used. A spray solvent of acetonitrile:water (9:1) was employed, with a spray voltage of 3.5 kV. The mass spectrometry parameters were optimized for each estrogen, using positive ion mode and specific parent and daughter ion transitions. Quantification was performed using Agilent MassHunter software, and calibration curves were generated. The limit of detection (LOD) was determined using a signal-to-noise ratio (S/N) of three. Recovery experiments were conducted using spiked blank samples to assess the accuracy of the method. Finally, the method's applicability was demonstrated by analyzing samples with a complex matrix representing typical cosmetic formulations.

The RPSI-MS method demonstrated excellent linearity (R² > 0.99) for all three estrogens across a concentration range of 0.002–1 µg/mL. The LOD for all three estrogens was 0.001 µg/mL. Recovery experiments showed values within the range of 80–111%, indicating good accuracy. The method exhibited significantly enhanced sensitivity compared to conventional PSI-MS; sensitivity improvements were 34,000-fold for estradiol, 80,000-fold for estriol, and 1400-fold for ethinylestradiol. The RPSI-MS method successfully quantified the target estrogens in cosmetic samples with a complex matrix, validating its applicability for real-world samples. The results are summarized in Table 1 and Table 2 in the original paper.

The significantly improved sensitivity of RPSI-MS compared to conventional PSI-MS highlights the effectiveness of the online derivatization strategy. The use of FluMP introduces a quaternary ammonium salt, significantly enhancing the ionization efficiency of the estrogens. The optimized protocol minimizes sample preparation steps, reducing analysis time and improving efficiency. The method's low LOD and good recovery demonstrate its suitability for accurate and reliable quantification of estrogens even at trace levels in complex cosmetic matrices. The successful application of the method to real-world samples with complex compositions validates its practical utility for cosmetic quality control and public health protection.

This study successfully developed a rapid, sensitive, and eco-friendly RPSI-MS method for detecting estrogens in cosmetics. The online derivatization approach significantly improved detection sensitivity compared to conventional PSI-MS. The method's simplicity, speed, and accuracy make it suitable for routine quality control testing. Future research could explore the application of this method to other matrices such as blood and urine, and investigate the use of more universal derivatization reagents to broaden the applicability of this approach to a wider range of compounds.

While the study demonstrated excellent sensitivity and accuracy, it is limited in scope by focusing on only three estrogens. Future studies should evaluate a wider range of estrogenic compounds. The specific composition of the complex matrix used in the recovery experiments may limit generalizability to all cosmetic formulations. Further investigations into potential matrix effects are recommended.