HPLC is commonly employed in the analysis of steroids as it provides an excellent separation and quantification tool. Regardless of the compound class, separation using the reversed phase mode is the method of choice in HPLC. Octadecyl silica (ODS or C₁₈) columns are commonly used as the stationary phase in reversed phase-HPLC. Other materials, such as C₈, C₂, phenyl, amino, and cyano phases can also be used to provide different degrees of selectivity. Selectivity depends also on the composition of the mobile phase. Methanol or acetonitrile are commonly used in combination with various percentages of water to prepare the mobile phase which can be employed in the isocratic or gradient mode. Column temperature, pH of the mobile phase and its modifiers are other parameters that can be used to optimize steroids separation. Using narrow and short columns can successfully decrease the amount of solvent needed and analysis time [4].

Prior to injecting steroid-containing samples on the HPLC, a sample preparation method is commonly employed to produce the required selectivity and sensitivity. Sample pretreatment is carried out through a process of sequential steps depending on the extracted steroid and sample type/matrix. For example, biological samples are often prepared by protein precipitation (PP) and enzyme hydrolysis in order to decrease the interferences from undesired compounds such as plasma proteins and to remove the polar groups added to steroids during metabolism. In addition to PP [9], solid phase extraction (SPE) [21,35] and liquid–liquid extraction (LLE) [36,37] are commonly used extraction techniques for the analysis of steroids. Due to the continuous need for faster and simpler sample preparation procedures, other methods such as solid phase microextraction (SPME) [38], liquid-phase microextraction (LPME) [37], turbulent flow chromatography (TFC) [39], dispersive liquid–liquid microextraction (DLLME) [37], molecularly imprinted solid-phase extraction (MISPE) [40,41], restricted access material (RAM) [4,9] are also used.

HPLC-FLD detection can be carried out through direct or indirect methods, depending on the analyte’s need for extraction or/and derivatization (Figure 2). Researchers seek to develop simple methods that do not require extraction or derivatization. For example, estrogens that are native fluorescents, and certain synthetic steroids, such as the anabolic steroid trenbolone, can fluoresce and, therefore, can be quantified without derivatization. Non-native steroids and non-aromatic compounds, on the other hand, show no fluorescence characteristics and must therefore be derivatized (labeled with fluorescent moieties) prior to analysis (Figure 2).

A good derivatization procedure should be simple with high yield and minimum side products and is performed under mild conditions to avoid decomposition of the target steroid [4]. Derivatization enhances the detection of steroids and can be done by either pre-column offline labeling or post-column online labeling (Figure 2), which ideally must be rapid. These procedures can, however, be accelerated by modifying the reaction’s temperature and pH or incorporating a catalyst. Figure 1 is a representation for the most common fluorescent derivatizing pathways and their targets in steroid analysis. There are several classes of fluorescent derivatizing agents including anthracenes, coumarins and phenanthrenes. The derivatization process depends on functionalization of specific groups that are targeted in steroids structure as well as the type of derivatization agent used. For example, steroids with primary alcohol moiety, e.g., cortisone, can be targeted with a fluorescent agent in a selective reaction thus differentiating them from steroids containing secondary and tertiary alcohol groups such as prednisolone. Similarly, other steroids, such as estrogens, have specific phenolic groups, which can also be targeted by other agents. Other functionalities that react with fluorescent derivatizing agents and employed in steroid quantification are ketone and ketolic groups as will be discussed later in this review. The various derivatization procedures reported in the literature used for steroid detection by HPLC-FLD including the reagents employed and conditions used in each procedure are summarized in Table 1.