Reactive Paper Spray Ionization Mass Spectrometry for Rapid Detection of Estrogens in Cosmetics

Hormones are vital signaling molecules with major physiological roles, including regulation of gene expression, reproductive physiology, pain pathways, immune function, and neuroprotection. Sex hormones (estrogens, androgens, progestogens) act at very low concentrations. Estrogens, which can lighten skin, reduce wrinkles, and slow skin aging, have been illicitly added to cosmetics. Prolonged use of estrogen-containing cosmetics can cause adverse effects, including metabolic dysfunctions, breast cancer, and endometrial hyperplasia. The European Union’s Cosmetics Regulation (EC No 1223/2009) prohibits the use of estrogens in cosmetics. Consequently, there is a critical need for simple, effective, and rapid analytical methods to detect and quantify estrogens in cosmetic products. This study develops a reactive paper spray ionization mass spectrometry (RPSI-MS) method using FluMP derivatization to rapidly detect estradiol, estriol, and ethinyloestradiol in cosmetics with minimal pretreatment.

Several analytical methods have been reported for estrogen detection in cosmetics. Tu et al. applied second-order calibration with excitation-emission matrix fluorescence to measure estriol and estrone in liquids. Hubinger used HPLC-UV to determine estriol, estradiol, estrone, and progesterone in cosmetics. De Orsi et al. used HPLC with diode-array and electrospray MS to analyze non-allowed substances in products for hair loss and hormone-related skin conditions. While effective, these approaches are time-consuming and require complex sample pretreatment. Ambient ionization mass spectrometry (AIMS) enables direct analysis under atmospheric conditions with minimal preprocessing; paper spray ionization (PSI-MS) is an AIMS technique combining advantages of AIMS and ESI. Reactive paper spray ionization (RPSI-MS) further integrates online derivatization to facilitate rapid qualitative and quantitative analysis of compounds with poor MS response in complex matrices. RPSI-MS has previously been used to detect quinones, aldehydes, and to probe reaction reactivity (e.g., Katritzky reaction). Building on this, FluMP derivatization has been shown to react with alcohols and phenols (including estrogens) and improve signal response in MS analyses.

Overview and derivatization strategy: The method employs RPSI-MS with online derivatization using 2-fluoro-1-methyl-pyridinium-p-toluene-sulfonate (FluMP), which reacts with primary/secondary alcohols and phenols to form quaternary ammonium derivatives, enhancing ionization and MS response. RPSI conditions accelerate derivatization during electrospray formation, improving efficiency relative to bulk reactions. Optimization of derivatization parameters: FluMP concentration/volume were optimized to ensure complete reaction without blocking the paper tip; 5 µL of 1 mg/mL FluMP was selected. Triethylamine provides basic conditions essential for derivatization. Best signal was achieved by adding triethylamine directly to the estrogen solution (v/v = 1:10) before loading onto paper; premixing triethylamine with FluMP or spray solvent, or preloading on the paper, gave poorer signals. Loading/drying time was evaluated: applying spray solvent immediately after loading the sample (without extended drying) yielded good signals; longer drying did not increase signal and could cause precipitation and hinder desorption. Quantification approach: Agilent MassHunter was used. Calibration curves were constructed by plotting analyte/IS response versus nominal concentrations. Lower limits of detection (LOD) used S/N = 3 criterion. Recovery study: A 50 mg blank cosmetic matrix was spiked with 500 µL linear standard at 0.01, 0.1, or 1 µg/mL, then 5 µL IS and 50 µL triethylamine were added, vortexed 10 s, and analyzed by RPSI-MS. Three replicates per level were run; RSDs were <10%. Complex matrix test: The method’s practicality was evaluated by spiking targets into a complex cosmetic-like matrix (water, butanediol, glyceryl polyacrylate, glycerinum, β-glucan, etc.) and quantifying by RPSI-MS. Reagents and materials: Estradiol, estriol, and ethinyloestradiol (National Institutes for Food and Drug Control, Beijing, China); acetonitrile (Sigma-Aldrich); FluMP (TCI, Shanghai); triethylamine (HUSHI, Shanghai). Estradiol valerate (200 µg/mL in ACN) was used as internal standard (IS). Instrumentation: Agilent 1290 HPLC coupled to 6470 triple quadrupole MS; HB-Z303-1AC high-voltage DC power supply; KQ-500DA ultrasonic cleaner; Whatman Grade 1 chromatographic paper. Data processed with Agilent MassHunter Workstation 10.1. Sample preparation: Mixed standard stock (10 µg/mL) prepared by dissolving ~0.2 mg each estradiol, estriol, ethinyloestradiol in 20 mL acetonitrile. Linear standards: 0.002, 0.01, 0.1, 0.5, 1 µg/mL in acetonitrile. IS: 200 µg/mL estradiol valerate. For each calibration solution: 500 µL linear standard + 5 µL IS + 50 µL triethylamine, mix. Sample solutions: dissolve 50 mg sample in 500 µL acetonitrile, add 5 µL IS and 50 µL triethylamine, mix. MS parameters: Paper triangles (Whatman Grade 1) 5 mm × 13 mm. Spray solvent: 20 µL acetonitrile:water (9:1). Spray voltage 3.5 kV. Tip-to-cone distance ~0.5 cm. Positive ion mode, MRM transitions: estradiol 364.2→128.1; estriol 380.2→128.1; ethinyloestradiol 388.2→128.1; collision energy 65 V for all. Gas and sheath temperatures 100 °C. Other parameters auto-optimized. RPSI-MS analysis: Spot 5 µL of 1 mg/mL FluMP on the paper and dry. Add 5 µL of standard or sample solution to the paper. Apply DC high voltage and then add 20 µL spray solvent to initiate spray desorption; acquire signals.

- The RPSI-MS method enabled rapid, minimal-preparation quantitation of estradiol, estriol, and ethinyloestradiol in cosmetics within minutes using online FluMP derivatization and estradiol valerate as IS. - Sensitivity improvement over conventional PSI-MS: estradiol ×34,000; estriol ×80,000; ethinyloestradiol ×1,400. - Linearity: 0.002–1 µg/mL for all three estrogens with R2 > 0.99 (estradiol R2 = 0.9917, estriol R2 = 0.9925, ethinyloestradiol R2 = 0.9960). Reported regression: estradiol Y = 1.405x − 0.003786; estriol Y = 1.430x − 0.005058; ethinyloestradiol Y = 0.8206x − 0.004691. - LOD: 0.001 µg/mL for each analyte (S/N = 3). - Recoveries (mean, n=3 per level; RSD <10%): • Estradiol: 105.8% (0.01 µg/mL), 95.3% (0.1 µg/mL), 85.7% (1 µg/mL) • Estriol: 110.0% (0.01 µg/mL), 97.0% (0.1 µg/mL), 83.0% (1 µg/mL) • Ethinyloestradiol: 84.0% (0.01 µg/mL), 80.5% (0.1 µg/mL), 85.0% (1 µg/mL) - Method performed accurately in a complex cosmetic-like matrix (water, butanediol, glyceryl polyacrylate, glycerinum, β-glucan, etc.), supporting applicability to real cosmetic samples.

The study addresses the need for rapid, straightforward detection of prohibited estrogens in cosmetics by leveraging RPSI-MS with online FluMP derivatization. Introducing a permanent positive charge via FluMP markedly enhanced ionization efficiency for phenolic/alcoholic estrogens, overcoming their typically poor MS response in PSI. Optimization of FluMP load, triethylamine addition strategy, and minimal drying enabled robust in situ derivatization directly on paper without extraction, simplifying workflow and reducing analysis time. The method showed excellent linearity (0.002–1 µg/mL), low LODs (0.001 µg/mL), acceptable recoveries (approximately 80–111%), and massive sensitivity gains versus PSI-MS. Demonstration in a complex matrix indicates resilience to common cosmetic excipients. Collectively, the findings show that RPSI-MS can deliver rapid, sensitive, and quantitative screening of estrogens in cosmetics with minimal pretreatment, supporting regulatory surveillance and quality control.

A reactive paper spray mass spectrometry (RPSI-MS) method using FluMP derivatization was developed for rapid analysis of estradiol, estriol, and ethinyloestradiol in liquid cosmetic samples. Compared to standard PSI, sensitivity increased by 34,000-fold (estradiol), 80,000-fold (estriol), and 1,400-fold (ethinyloestradiol). The method provided good linearity over 0.002–1 µg/mL with LODs of 0.001 µg/mL and acceptable recoveries. RPSI-MS is well suited for low-response compounds in MS, and future work may develop more universal derivatization reagents and extend applications to biofluids (e.g., blood or urine). Overall, RPSI-MS shows strong potential for rapid detection of compounds with poor response in conventional PSI-MS.

- The approach depends on suitable derivatization chemistry; there is no universal derivatization reagent for all functional groups, and selecting effective reagents remains challenging. - Excess derivatization reagent (FluMP) can clog the paper tip; drying time can lead to precipitation and hinder desorption, requiring careful optimization. - Validation focused on three estrogens and cosmetic matrices; broader analyte classes and diverse real-world products warrant further evaluation. - Although proposed for future use in biological matrices, such applications were not demonstrated here.