Determination of 42 antimicrobials in disinfection products by dispersive solid phase extraction-high performance liquid chromatography-tandem mass spectrometry

Antimicrobials inhibit the growth and reproduction of microorganisms thereby alleviating skin and other problems caused by microorganisms. Antimicrobials are classified into different categories, including antibacterials, antifungals, and antivirals, among others, and include azoles, sulfonamides, tetracyclines, quinolones, and many other classes of synthetic and natural compound. The inappropriate or excessive use of antimicrobials can damage skin and other human organs and increase antimicrobial resistance. Relevant regulations and standards clearly state that antimicrobials are prohibited for use as ingredients in disinfection products. However, since antimicrobials enhance the disinfection or antibacterial effect of a product, with a significant short-term effect, antimicrobials are occasionally illegally added to disinfectant products, including those intended for human use. Therefore, establishing testing methods that provide technical support for enforcing regulations is an urgent objective. Herein, a method was established for the analysis of 42 antimicrobials in disinfection products, that is applicable to common types of disinfection-product matrix, including creams, gels, and aqueous solutions, using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) combined with dispersive solid phase extraction. The 42 antimicrobials comprise antibacterials, antifungals, and antivirals, and include seven sulfonamides, ten quinolones, three lincosamides, five tetracyclines, three macrolides, eight azoles, three purine nucleoside analogs, one furan, one nonpolyene antifungal, and one steroid. Briefly, 0.2 g of a sample was first dispersed in 2 mL of water and then extracted with 10 mL of 0.5% formic acid in acetonitrile, with 3 g of anhydrous Na₂SO₄ added to remove water. After centrifugation, 5 mL of the supernatant was cleaned using dispersive solid phase extraction with EMR-Lipid as the adsorbent. Lipids, waxes, surfactants, and moisturizing lubricants are commonly used as cream and gel matrices. Matrix substances containing long carbon chains dissolved in acetonitrile were removed using the EMR-Lipid adsorbent. Target analytes were separated on a Poroshell 120 EC-C18 analytical chromatography column (150 mm×3.0 mm, 2.7 μm), with 0.1% formic acid in acetonitrile and 0.1% formic acid aqueous solution used as mobile phases under gradient-elution conditions. The target analytes in the test solution were detected in positive ionization (ESI⁺) and multi-reaction-monitoring (MRM) modes. Analytes were characterized in terms of their retention times and selected ions, and quantified using the external-standard method. The main factors affecting method response, recovery, and sensitivity, such as the extraction method and solvent, purification method and adsorbent, mobile phase, and MS conditions, were examined during sample pretreatment and instrumental analysis. The 42 antimicrobials were effectively separated under the optimized experimental conditions; the target compounds exhibited linear working curves in the 0.25-5.0 mg/kg concentration range, with correlation coefficients (<i>r</i>) greater than 0.99. Limits of detection (LODs) for the 42 antimicrobials were determined from the signal-to-noise ratios (<i>S/N</i>) of their chromatographic peaks. LODs of 0.03-0.10 mg/kg were determined for the three matrices using 0.2-g samples and 10-mL test solution. Recoveries of 80.3-109.8%, with relative standard deviations (RSDs) of less than 9.8%, were obtained by determining three levels of each target analyte added to the three blank matrices; this process was repeated for six parallel samples. The developed method was used to analyze antimicrobials in commercially available disinfection products, with two sample batches testing positive. The established method is simple, accurate, precise, and suitable for the rapid screening and quantification of antimicrobials in disinfection products. This study provides powerful technical support for regulating the illegal addition of related antimicrobials to disinfection products.