## 1 Introduction
Immunological techniques offer some advantages with respect to the traditional chromatography-based methods, such as easier and faster protocol, as well as suitability to analyze a large number of samples. The specificity of the antigen-antibody reaction makes this approach ideal for the detection of small traces of the target analyte in very complex matrices [47]. Furthermore, immunological methods can be performed easily and rapidly (the tests are generally completed in a few hours) and the protocol is suitable for complete automation and high-throughput screening. Finally, execution of immunoassay requires an aqueous environment, avoiding the use of organic solvents and toxic chemicals and thus allowing for working with biocompatible and green procedures. There are different formats in which immunological methods can be implemented, but the most used for routinary applications is the ELISA. An enzyme (such as horseradish peroxidase, HRP) is used as a label in order to catalyze a reaction in the presence of a specific enzyme substrate in order to generate a signal that is measured for quantitative detection of the target analyte. The assay is performed in a microwell plate platform, and a multistep procedure comprising the addition of the sample and of the immunoreagents followed by several washing steps is required. Finally, the addition of the enzymatic substrate allows for the development of the analytical signal, which can be detected by exploiting a multi-well plate reader. Depending on the nature of the enzymatic substrate, it is possible to exploit different detection principles (e.g., colorimetric, chemiluminescent, or electrochemiluminescence detection). The traditional reference techniques, such as HPLC, GC coupled with MS, and immunoassays, show a limitation in sensitivity. Indeed, the limit of detection (LOD) and quantification (LOQ) are generally higher than the ones offered by instrumental-based techniques. Another problem is represented by the cross-reactivity phenomenon, which implies that the antibody recognizes the target antigen and similar species, leading to a lack of specificity. However, immunoassays can be very useful as preliminary screening methods to be applied in series on a great number of samples in order to identify batches suspected of adulteration, which can then be subjected to further analysis [48]. In this study, an HPLC-MS method was developed for the simultaneous determination of eight corticosteroids (Figure 1): betamethasone valerate (BM 17-V), beclomethasone (BC), beclomethasone dipropionate (BCDP), methylprednisolone (MP), budesonide (BD), flunisolide (FN), flumenthasone (FM), and dexamethasone (DM) in cosmetic formulations without any sample pre-treatment. The method was validated in terms of linearity, sensitivity, accuracy, precision, and matrix effect on three cosmetic products (two creams and one serum) available on the market. Moreover, an alternative method based on ELISA was employed to detect the aforementioned corticosteroids. This technique was adopted to simplify the analysis of cosmetic formulations to determine the presence of glucocorticoids without the need for a chromatography instrument, which might not be available in every cosmetic lab. The ELISA was developed starting from a commercial kit for the measurement of flumenthasone in plasma, extending its applicability to cosmetic matrices and to a multi-target screening (5 corticosteroids: BM 17-V, BC, MP, FM, and DM). The ELISA protocol in use was optimized and validated on two steroid-free commercial cosmetic products (cream and serum). For the first time, an ELISA was validated for the preliminary detection of a group of corticosteroids at the same time. This method can be of great support for the rapid control of samples suspected of adulteration with glucocorticoids in order to increase the action of the product claim.