High Performance Liquid Chromatography (HPLC) with Fluorescence Detection for Quantification of Steroids in Clinical, Pharmaceutical, and Environmental Samples: A Review

Steroids are compounds widely available in nature and synthesized for therapeutic and medical purposes. Although several analytical techniques are available for the quantification of steroids, their analysis is challenging due to their low levels and complex matrices of the samples. The efficiency and quick separation of the HPLC combined with the sensitivity, selectivity, simplicity, and cost-efficiency of fluorescence, make HPLC coupled to fluorescence detection (HPLC-FLD) an ideal tool for routine measurement and detection of steroids. In this review, we covered HPLC-FLD methods reported in the literature for the steroids quantification in clinical, pharmaceutical, and environmental applications, focusing on the various approaches of fluorescent derivatization. The aspects related to analytical methodology including sample preparation, derivatization reagents, and chromatographic conditions will be discussed.


Introduction
Steroids are biologically active molecules that are available in many natural sources such as, animals, plants and fungi, in addition to being manufactured as therapeutics. Steroids share a common four-fused ring core consisting of 17 carbon atoms and vary according to functional groups attached to the core or side chains. Naturally secreted steroids are classified into steroid hormones (sex hormones, mineralo-and glucocorticoids, and anabolic steroids) and cholesterol. These compounds travel the bloodstream bound specifically or non-specifically to plasma proteins, such as albumin and binding globulins [1]. Steroids are key players in many biological, physiological, and pathological events, which are mediated after binding to their cognate tissue receptors. Most prominently, natural steroids play major roles in metabolism, signaling, immunity, reproduction, and salt-retaining activity. On the other hand, many synthetic steroids have been manufactured to mimic natural steroidal activity and have been approved to treat a wide variety of diseases including anti-inflammatory; arthritis, autoimmune disease, asthma and chronic obstructive pulmonary diseases [2,3].
Because steroids can produce biological action at very low concentrations, analytical methods must be very sensitive and precise particularly when quantifying steroid levels in biosamples. Due to the complex nature matrices of most steroid samples, the methods should also be selective to reliably quantify target steroids and resolve them from other similar endogenous compounds and interferences, and to allow for simpler extraction and pre-treatment prior to analysis. This is especially important since sample pre-treatment Commonly used methods in the analysis of steroids include immuno-assays [5,6], gas chromatography-mass spectrometry (GC-MS) [7], high-performance liquid chromatography mass spectrometry (HPLC-MS or MS/MS) [4,8], capillary electrophoresis (CE) [9], and HPLC coupled with UV [10][11][12][13] or fluorescence detection (FLD) [14]. Mass spectrometry (MS) and FLD are relevant to steroid analysis due to their inherited high sensitivity detection and low detection limit. Although both usually require incorporating a derivatization step due to weak ionization of steroids in MS and lack of fluorophores, FLD is considered a simpler and low capital cost technique.
Levels of steroids are required to be continuously monitored since any increase or decrease may be associated with major consequences on health and environment. Therefore, steroid quantification has been gaining increasing importance in disease diagnosis (e.g., increased levels of cortisol in stress and Cushing's syndrome) [15], contamination assessment of water and food samples [16][17][18][19][20][21], detection of anabolic steroids among sportsmen due to doping [22][23][24][25][26], and detection of growth promoting steroids in animal tissue [18,25,[27][28][29][30][31][32][33]. While there are several reviews on steroid quantification by HPLC methods coupled to MS and UV [8,34], to the best of our knowledge no similar reports summarizing data on HPLC-FLD methods exist. This review will discuss the applications of HPLC-FLD methods for quantifying steroids in clinical, pharmaceutical, and environmental samples. Focus will be given to the various derivatization techniques reported in the literature to introduce fluorophores to target steroids and accompanying sample preparation procedures.

HPLC-FLD Methods
HPLC is commonly employed in the analysis of steroids as it provides an excellent Commonly used methods in the analysis of steroids include immuno-assays [5,6], gas chromatography-mass spectrometry (GC-MS) [7], high-performance liquid chromatography mass spectrometry (HPLC-MS or MS/MS) [4,8], capillary electrophoresis (CE) [9], and HPLC coupled with UV [10][11][12][13] or fluorescence detection (FLD) [14]. Mass spectrometry (MS) and FLD are relevant to steroid analysis due to their inherited high sensitivity detection and low detection limit. Although both usually require incorporating a derivatization step due to weak ionization of steroids in MS and lack of fluorophores, FLD is considered a simpler and low capital cost technique.
Levels of steroids are required to be continuously monitored since any increase or decrease may be associated with major consequences on health and environment. Therefore, steroid quantification has been gaining increasing importance in disease diagnosis (e.g., increased levels of cortisol in stress and Cushing's syndrome) [15], contamination assessment of water and food samples [16][17][18][19][20][21], detection of anabolic steroids among sportsmen due to doping [22][23][24][25][26], and detection of growth promoting steroids in animal tissue [18,25,[27][28][29][30][31][32][33]. While there are several reviews on steroid quantification by HPLC methods coupled to MS and UV [8,34], to the best of our knowledge no similar reports summarizing data on HPLC-FLD methods exist. This review will discuss the applications of HPLC-FLD methods for quantifying steroids in clinical, pharmaceutical, and environmental samples. Focus will be given to the various derivatization techniques reported in the literature to introduce fluorophores to target steroids and accompanying sample preparation procedures.

HPLC-FLD Methods
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 C 18 ) columns are commonly used as the stationary phase in reversed phase-HPLC. Other materials, such as C 8 , C 2 , 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. Table 1. Derivatization reagents and conditions used in steroid quantification by HPLC-FLD.

Reagent Target Steroid Sample Preparation and Derivatization Conditions Reference
p-Nitrobenzoyl chloride 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.     Dansyl hydrazine F, Butane acid-(5-androsten-17one-3beta-ol)diester (A1998) [69], alfaxalone and pregnanolone [70] Sample was extracted by LLE. Extracts reacted with dansyl hydrazine in organic solvent for 30 min at RT in acidic medium.  [70] F, Butane acid-(5-androsten-17one-3beta-ol)-diester (A1998) [69], alfaxalone and pregnanolone [70] Sample was extracted by LLE. Extracts reacted with dansyl hydrazine in organic solvent for 30 min at RT in acidic medium. Kawasaki et al. 1979 [71] Visser et al. 2000 [69] Peng et al. 2007 [70]  The samples were extracted by LLE. BQEIC in a solvent of DMAP at 80 °C for 60 min. Finally, the mixture was cooled to RT and diluted with acetonitrile.

Clinical Applications of HPLC-FLD Techniques
Quantification of steroids plays an important role in the diagnosis and treatment of endocrine disorders. Diseases, such as cancer, metabolic syndromes, and neurodegenerative diseases, are associated with abnormalities in the endocrine system. Steroids also play an important role in biochemical processes, such as aging, reproduction, and metabolic pathways [76]. Furthermore, disturbances in the endocrine system due to EDC from environment and food are becoming a major clinical concern. HPLC-FLD has been employed in many research studies, both in humans and animal models, to quantify the various types of steroids and investigate their role in diseases and clinical conditions.

Detection of Glucocorticosteroids
Glucocorticosteroids are widely recognized as markers for adrenal activity. Cortisol can reflect the short-term changes in the activity of the hypothalamic-pituitary-adrenocortical axis (HPA axis), making it a valuable surrogate marker for stress and glucose metabolism [56].
Many methods have been developed to detect unconjugated cortisol levels in various 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).

Clinical Applications of HPLC-FLD Techniques
Quantification of steroids plays an important role in the diagnosis and treatment of endocrine disorders. Diseases, such as cancer, metabolic syndromes, and neurodegenerative diseases, are associated with abnormalities in the endocrine system. Steroids also play an important role in biochemical processes, such as aging, reproduction, and metabolic pathways [76]. Furthermore, disturbances in the endocrine system due to EDC from environment and food are becoming a major clinical concern. HPLC-FLD has been employed in many research studies, both in humans and animal models, to quantify the various types of steroids and investigate their role in diseases and clinical conditions.

Detection of Glucocorticosteroids
Glucocorticosteroids are widely recognized as markers for adrenal activity. Cortisol can reflect the short-term changes in the activity of the hypothalamic-pituitaryadrenocortical axis (HPA axis), making it a valuable surrogate marker for stress and glucose metabolism [56].
Many methods have been developed to detect unconjugated cortisol levels in various biological samples. However, the use of certain samples, such as urine, may not be recommended as these samples may contain many interfering substances thus complicating steroids extraction procedure [55]. In addition, the concentrations of corticosteroids in urine are low compared with those found in plasma, which makes the latter a better medium for measurement. Steroids have been measured by HPLC-FLD methods for various clinical purposes, such as investigating disease courses, pathogenesis, and metabolic processes [15].
The detection of corticosteroids in biological samples can be done directly depending on fluorescence signal enhancement upon the treatment of samples with the deproteinizing agent (ethanol) and sulfuric acid ( Figure 3). A study to quantify corticosteroids in serum samples was performed using C 18 analytical column after treatment with ethanol and sulfuric acid [53]. Cortisol, testosterone and corticosterone emitted fluorescence, with LOD for cortisol = 0.3 pg/dL (S/N = 3). In another study, the same group measured cortisol in urine samples producing a LOD = 0.26 pg/dL (S/N = 3) [54]. Sudo et al. used post-column derivatization with sulfuric acid to measure cortisol and corticosterone in rat urine (LOD was 0.5 pmol for corticosterone at (S/N = 5)) [55]. They analyzed other corticoids, such as prednisolone, 6α-methylprednisolone, dexamethasone, and betamethasone, but with lower sensitivity. Moreover, ethyl acetate was used for extraction after sulfuric acid hydrolysis to prevent the acid from entering the HPLC system. It was noticed that the fluorescence intensities were dependent on the reactor temperature and sulfuric acid [55,77]. Since cortisol levels in hair might be used as a biomarker of chronic stress, a study developed a HPLC-FLD method to measure cortisol distribution in human hair samples. The method has achieved equal precision to mass spectrometry. Samples were prepared by pulverization and incubation in 0.1 M HCl, and extraction with ethyl acetate followed by derivatization with sulfuric acid. A detection limit of 1 pg/mg was achieved [56].
Derivatization with fluorophore-containing reagents was employed in many studies to detect and quantify corticosteroids in serum and urine samples. The derivatization process depends on functionalization of specific groups in corticosteroids structure ( Figure  1). The primary alcohol functionality can be esterified through its reaction with either 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole (CDB) (Figure 4), 9-anthroyl nitrile (9-AN) ( Figure 5), or 1-anthroyl nitrile (1-AN) ( Figure 5). These derivatization processes proved to be selective as neither secondary alcohol, nor tertiary alcohol can react with fluorophore-containing reagents ( Figure 5). Since cortisol levels in hair might be used as a biomarker of chronic stress, a study developed a HPLC-FLD method to measure cortisol distribution in human hair samples. The method has achieved equal precision to mass spectrometry. Samples were prepared by pulverization and incubation in 0.1 M HCl, and extraction with ethyl acetate followed by derivatization with sulfuric acid. A detection limit of 1 pg/mg was achieved [56].
Derivatization with fluorophore-containing reagents was employed in many studies to detect and quantify corticosteroids in serum and urine samples. The derivatization process depends on functionalization of specific groups in corticosteroids structure ( Figure 1). The primary alcohol functionality can be esterified through its reaction with either 2-(4carboxyphenyl)-5,6-dimethylbenzimidazole (CDB) (Figure 4), 9-anthroyl nitrile (9-AN) ( Figure 5), or 1-anthroyl nitrile (1-AN) ( Figure 5). These derivatization processes proved to be selective as neither secondary alcohol, nor tertiary alcohol can react with fluorophorecontaining reagents ( Figure 5).   The derivatization through esterification of corticosteroid primary alcohol functionality with CDB was studied by Katayama [78]. The results showed that the detector response to secondary alcohols was less than one fiftieth that of the primary alcohol. However, certain secondary alcohols in steroids, such as prednisolone acetate and testosterone, and tertiary alcohols, showed no reaction. Later, Katayama used human plasma to detect eight corticosteroids by derivatization with CDB to their esters in acetonitrile. The esters were separated on a reversed-phase column (Zorbax ODS) with water:methanol (25:75, v/v) containing 5 mmol/L tetramethylammonium hydrogen sulfate as a mobile phase. The LOD ranged between 0.06 and 0.3 pg per 100 μL of plasma (S/N = 3) [58].
The use of 9-AN as a derivatizing agent for corticosteroids was reported in several studies. Neufled et al. used 9-AN to derivatize the primary alcohol in C21 corticosteroids. The reaction was carried out at 45 °C for 2 h and the fluorescent derivatives were separated on silica stationary phase with a mixture of 2-propanol and hexane as a mobile phase in the gradient mode. The low temperature in derivatization prevented the thermal degradation of corticosteroids and avoided reaction with secondary hydroxyl groups [46,50].
Measuring endogenous glucocorticoids and their metabolites is important as disturbances in the enzyme responsible for their metabolism can cause hypertension. One study described an HPLC-FLD method using 9-AN derivatization for the determination of cortisol, cortisone, and their tetrahydro-and allo-tetrahydro-metabolites in plasma and urine [45]. Following extraction with dichloromethane and SPE, 9-AN was used to derivatize the steroids in the samples to their fluorescent products. LOD (S/N = 3:1) achieved with this method was 3.0 ng/mL for all analytes. Similarly, Shibata et al. employed 9-AN for fluorescent derivatization and developed a method to investigate cortisone levels in plasma and urine samples of renal transplant patients who received prednisolone [47]. Cortisol, cortisone, prednisolone, prednisone, 6β-hydroxycortisol, and 6β-hydroxyprednisolone were derivatized to their fluorescent esters after extraction with ethyl acetate ( Figure 5).
The 6β-hydroxycortisone was used as an internal standard. LODs achieved for cortisol, cortisone, prednisolone and prednisone in plasma or urine were 0.1 ng/mL, while The derivatization through esterification of corticosteroid primary alcohol functionality with CDB was studied by Katayama et al. [78]. The results showed that the detector response to secondary alcohols was less than one fiftieth that of the primary alcohol. However, certain secondary alcohols in steroids, such as prednisolone acetate and testosterone, and tertiary alcohols, showed no reaction. Later, Katayama et al. used human plasma to detect eight corticosteroids by derivatization with CDB to their esters in acetonitrile. The esters were separated on a reversed-phase column (Zorbax ODS) with water:methanol (25:75, v/v) containing 5 mmol/L tetramethylammonium hydrogen sulfate as a mobile phase. The LOD ranged between 0.06 and 0.3 pg per 100 µL of plasma (S/N = 3) [58].
The use of 9-AN as a derivatizing agent for corticosteroids was reported in several studies. Neufled et al. used 9-AN to derivatize the primary alcohol in C 21 corticosteroids. The reaction was carried out at 45 • C for 2 h and the fluorescent derivatives were separated on silica stationary phase with a mixture of 2-propanol and hexane as a mobile phase in the gradient mode. The low temperature in derivatization prevented the thermal degradation of corticosteroids and avoided reaction with secondary hydroxyl groups [46,50].
Measuring endogenous glucocorticoids and their metabolites is important as disturbances in the enzyme responsible for their metabolism can cause hypertension. One study described an HPLC-FLD method using 9-AN derivatization for the determination of cortisol, cortisone, and their tetrahydro-and allo-tetrahydro-metabolites in plasma and urine [45]. Following extraction with dichloromethane and SPE, 9-AN was used to derivatize the steroids in the samples to their fluorescent products. LOD (S/N = 3:1) achieved with this method was 3.0 ng/mL for all analytes. Similarly, Shibata et al. employed 9-AN for fluorescent derivatization and developed a method to investigate cortisone levels in plasma and urine samples of renal transplant patients who received prednisolone [47]. Cortisol, cortisone, prednisolone, prednisone, 6β-hydroxycortisol, and 6β-hydroxyprednisolone were derivatized to their fluorescent esters after extraction with ethyl acetate ( Figure 5). The 6β-hydroxycortisone was used as an internal standard. LODs achieved for cortisol, cortisone, prednisolone and prednisone in plasma or urine were 0.1 ng/mL, while those for 6β-hydroxycortisol and 6β-hydroxyprednisolone in plasma or urine were 0.5 ng/mL. Similar to 9-AN, fluorescent derivatization can be achieved using 1-AN. A study described the use of 1-AN to derivatize 18-oxygenated corticosteroids: 18-hydroxycortisol, 18-hydroxycortisone and 18-oxocortisol in human urine into their fluorescent 21-anthroyl esters [43] (Figure 6). A mixture of ether and dichloromethane was used to extract the steroids, and the anthroyl derivatives were enriched by SPE using a CN cartridge column. The LOD was 0.1 pmol (S/N = 5). those for 6β-hydroxycortisol and 6β-hydroxyprednisolone in plasma or urine were 0.5 ng/mL. Similar to 9-AN, fluorescent derivatization can be achieved using 1-AN. A study described the use of 1-AN to derivatize 18-oxygenated corticosteroids: 18-hydroxycortisol, 18-hydroxycortisone and 18-oxocortisol in human urine into their fluorescent 21-anthroyl esters [43] (Figure 6). A mixture of ether and dichloromethane was used to extract the steroids, and the anthroyl derivatives were enriched by SPE using a CN cartridge column. The LOD was 0.1 pmol (S/N = 5). Fluorescent derivatization of corticosteroids can also be carried out using dansyl hydrazine targeting the carbonyl groups (Figure 7). In a study to determine corticosteroids in human plasma and urine samples were derivatized by dansyl hydrazine and quantified by HPLC-FLD, following extraction with methylene chloride. The linearity range of the method was between 0.5 and 60 ng of cortisol, proving it to be suitable for the routine analysis of cortisol in plasma and urine [71]. Fluorescent derivatization of corticosteroids can also be carried out using dansyl hydrazine targeting the carbonyl groups (Figure 7). In a study to determine corticosteroids in human plasma and urine samples were derivatized by dansyl hydrazine and quantified by HPLC-FLD, following extraction with methylene chloride. The linearity range of the method was between 0.5 and 60 ng of cortisol, proving it to be suitable for the routine analysis of cortisol in plasma and urine [71]. The detection of corticosteroids with ketolic groups in urine is key part in diagnostic procedure of Cushing's syndrome. Ketol-containing corticosteroids, such as 17-hydroxycorticosteroid (breakdown product of cortisol), are usually excreted in urine as tetrahydro form making their detection using UV absorption at 240 nm not possible. To detect these steroids in urine, derivatization with amidine-containing compounds was used yielding fluorescent compounds [60] (Figure 8). Using this approach, the best sensitivity was achieved for cortisol. The detection of corticosteroids with ketolic groups in urine is key part in diagnostic procedure of Cushing's syndrome. Ketol-containing corticosteroids, such as 17hydroxycorticosteroid (breakdown product of cortisol), are usually excreted in urine as tetrahydro form making their detection using UV absorption at 240 nm not possible. To detect these steroids in urine, derivatization with amidine-containing compounds was used yielding fluorescent compounds [60] (Figure 8). Using this approach, the best sensitivity was achieved for cortisol. The utilization of ketolic groups was also performed by Yamaguchi et al. to detect and quantify corticosteroids in urine after their conversion into fluorescent quinoxalines (Figure 9) [67]. They detected nineteen 21-hydroxycorticosteroids in human urine samples achieving LOD = 0.14-29.4 pmol/50 μL injection volume (S/N = 3). In another study by the same group, prednisolone and prednisone were quantified in plasma samples following similar derivatization procedure and analyzed by reversed-phase liquid chromatography with isocratic elution. The LOD of prednisolone and prednisone was 3 ng/mL in plasma (S/N = 3) [65]. The utilization of ketolic groups was also performed by Yamaguchi et al. to detect and quantify corticosteroids in urine after their conversion into fluorescent quinoxalines (Figure 9) [67]. They detected nineteen 21-hydroxycorticosteroids in human urine samples achieving LOD = 0.14-29.4 pmol/50 µL injection volume (S/N = 3). In another study by the same group, prednisolone and prednisone were quantified in plasma samples following similar derivatization procedure and analyzed by reversed-phase liquid chromatography with isocratic elution. The LOD of prednisolone and prednisone was 3 ng/mL in plasma (S/N = 3) [65]. Cholesterol quantification is commonly required in the analysis of biosamples. A study described a simple and sensitive method for the determination of cholesterol and phytosterols, such as β-sitosterol in biosamples (e.g., saliva and urine matrices) and food samples (cow and soybean milk) after derivatization with naproxen acyl chloride in toluene ( Figure 10). The method employed a C8 column with a mixture of methanol, isopropanol, and water, achieving a LOD of about 25 nM (S/N = 3). The analysis of cholesterol and sitosterol is usually time-consuming, but using the method, a relatively short period of time was required since solvent concentration, evaporation, and replacement steps were not necessary [79]. Cholesterol quantification is commonly required in the analysis of biosamples. A study described a simple and sensitive method for the determination of cholesterol and phytosterols, such as β-sitosterol in biosamples (e.g., saliva and urine matrices) and food samples (cow and soybean milk) after derivatization with naproxen acyl chloride in toluene ( Figure 10). The method employed a C 8 column with a mixture of methanol, isopropanol, and water, achieving a LOD of about 25 nM (S/N = 3). The analysis of cholesterol and sitosterol is usually time-consuming, but using the method, a relatively short period of time was required since solvent concentration, evaporation, and replacement steps were not necessary [79]. Free 7α-hydroxycholesterol (7-HC) levels in human serum were reported to be a good indicator for bile acid synthesis. An HPLC-FLD method was developed by Saisho et al. to quantify 7-HC levels in dog plasma, with the purpose of studying the effect of cholestyramine on plasma levels of 7-HC. 7-HC was converted to its fluorescence derivative, by two procedures, after being extracted and then purified [57]. The two derivatization reagents used were 1-AN and 7-methoxycoumarin-3-carbonyl azide (MC-CON3). The MC-CON3 derivatization resulted in higher fluorescence intensity compared to 1-AN Free 7α-hydroxycholesterol (7-HC) levels in human serum were reported to be a good indicator for bile acid synthesis. An HPLC-FLD method was developed by Saisho et al. to quantify 7-HC levels in dog plasma, with the purpose of studying the effect of cholestyramine on plasma levels of 7-HC. 7-HC was converted to its fluorescence derivative, by two procedures, after being extracted and then purified [57]. The two derivatization reagents used were 1-AN and 7-methoxycoumarin-3-carbonyl azide (MC-CON 3 ). The MC-CON 3 derivatization resulted in higher fluorescence intensity compared to 1-AN route. 1-AN produced only a C-3 fluorescent derivative due to the bulky anthracene group and steric hindrance around the C-7 position. In comparison, MC-CON 3 yielded a double coumarin derivative at the C-3 and C-7 positions ( Figure 11) producing a LOD of 4 pg (S/N = 5).

Detection of Steroid Hormones
Detection of steroid hormones and their metabolites are important in diagnosis of metabolic diseases. Several HPLC-FLD methods were described for the detection of steroid hormones. A study developed a method for monitoring progesterone and 17-hydroxyprogesterone in the serum from pregnant women. The quantification employed fluorescent derivatization using 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionohydrazide (BODIPY TM FL hydrazide) [71] (Figure 12). The derivatization was carried out in ethanol at room temperature (about 22 °C) for 15 h. This derivatization method was reported to be 50 times faster than that using dansyl hydrazine. The LODs for progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone and 17-methyltestosterone were in the range of 550-3700 fmol per 10 μL injection (S/N = 5).

Detection of Steroid Hormones
Detection of steroid hormones and their metabolites are important in diagnosis of metabolic diseases. Several HPLC-FLD methods were described for the detection of steroid hormones. A study developed a method for monitoring progesterone and 17hydroxyprogesterone in the serum from pregnant women. The quantification employed fluorescent derivatization using 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionohydrazide (BODIPY TM FL hydrazide) [71] (Figure 12). The derivatization was carried out in ethanol at room temperature (about 22 • C) for 15 h. This derivatization method was reported to be 50 times faster than that using dansyl hydrazine. The LODs for progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone and 17-methyltestosterone were in the range of 550-3700 fmol per 10 µL injection (S/N = 5). The derivatization reagent 1-AN was used by Shimada et al. to determine the endogenous steroid pregnenolone in Wistar and Sprague-Dawley rat brain samples [46]. The samples were homogenized in isotonic saline then deproteinized with methanol, before subjected to sequential steps of extraction and derivatization. The samples were derivatized with 1-AN ( Figure 13) and the excess reagent was removed by purification, carried The derivatization reagent 1-AN was used by Shimada et al. to determine the endogenous steroid pregnenolone in Wistar and Sprague-Dawley rat brain samples [46]. The samples were homogenized in isotonic saline then deproteinized with methanol, before subjected to sequential steps of extraction and derivatization. The samples were derivatized with 1-AN ( Figure 13) and the excess reagent was removed by purification, carried out on two successive silica gel columns. The 3β-hydroxy-16-methylpregna-5,16-dien-20-one was used as an internal standard. Several steroids in animal tissue were detected in another study using ultraviolet, fluorometric, and electrochemical detectors [80]. The study demonstrated the selectivity of FLD in determination of estradiol by exploiting its fluorescence emission, although it was eluted with the same fraction containing other steroids and their respective metabolites. Extraction with methanol allowed the separation of acidic corticosteroids (diethylstilbestrol, estradiol, zeranol/zearalenone, and their metabolites) from their neutral anabolic counterparts (testosterone, trenbolone and progesterone).
Urine is the best source for the estimation of estrogen concentrations, since they are primarily excreted renally as glucuronides and sulfates. Previous studies used urine samples for the determination of estrogen levels in their studies, albeit with different extraction methods [42,81]. Mao et al. used chemical hydrolysis in methanol and concentrated hydrochloric acid at 70 °C for 1 h to release the conjugation before SPE sample preparation, whereas Kumar et al. used a fabric phase sorptive extraction (FBSE) procedure, which offered shorter sample preparation times and 400 times higher sorbent loading. This method achieved a lower detection limit and analysis time, proving to be greener and more economical. The LODs for β-estradiol, 17α-ethinylestradiol, and bisphenol A were 20 pg/mL, 36 pg/mL, and 42 pg/mL, respectively.
In another study, Mao used p-nitrobenzoyl chloride at 25 °C as a derivatization reagent, which can easily react with the hydroxyl and phenolic hydroxyl groups of organic chemicals ( Figure 14) without the need for a catalyst [42]. Moreover, 4-nonylphenol, bisphenol A (BPA), 17α-ethinylestradiol, and three endogenic estrogens, including 17α-estradiol, 17β-estradiol, and estriol, were determined in urine samples collected from 20 healthy volunteers. Samples were hydrolyzed with HCl and subjected to SPE purification. Separation was performed on a C18 column with gradient elution using acetonitrile and water as a mobile phase. The LODs of the method were 2.7 μg/L for BPA and 17β-estradiol, 2.9 μg/L for 4-nonylphenol, 4.6 μg/L for 17α-estradiol and 17α-ethinylestradiol, and 8.3 μg/L for estriol. Several steroids in animal tissue were detected in another study using ultraviolet, fluorometric, and electrochemical detectors [80]. The study demonstrated the selectivity of FLD in determination of estradiol by exploiting its fluorescence emission, although it was eluted with the same fraction containing other steroids and their respective metabolites. Extraction with methanol allowed the separation of acidic corticosteroids (diethylstilbestrol, estradiol, zeranol/zearalenone, and their metabolites) from their neutral anabolic counterparts (testosterone, trenbolone and progesterone).
Urine is the best source for the estimation of estrogen concentrations, since they are primarily excreted renally as glucuronides and sulfates. Previous studies used urine samples for the determination of estrogen levels in their studies, albeit with different extraction methods [42,81]. Mao et al. used chemical hydrolysis in methanol and concentrated hydrochloric acid at 70 • C for 1 h to release the conjugation before SPE sample preparation, whereas Kumar et al. used a fabric phase sorptive extraction (FBSE) procedure, which offered shorter sample preparation times and 400 times higher sorbent loading. This method achieved a lower detection limit and analysis time, proving to be greener and more economical. The LODs for β-estradiol, 17α-ethinylestradiol, and bisphenol A were 20 pg/mL, 36 pg/mL, and 42 pg/mL, respectively.
In another study, Mao et al. used p-nitrobenzoyl chloride at 25 • C as a derivatization reagent, which can easily react with the hydroxyl and phenolic hydroxyl groups of organic chemicals ( Figure 14) without the need for a catalyst [42]. Moreover, 4-nonylphenol, bisphenol A (BPA), 17α-ethinylestradiol, and three endogenic estrogens, including 17αestradiol, 17β-estradiol, and estriol, were determined in urine samples collected from 20 healthy volunteers. Samples were hydrolyzed with HCl and subjected to SPE purification. Separation was performed on a C 18 column with gradient elution using acetonitrile and water as a mobile phase. The LODs of the method were 2.7 µg/L for BPA and 17β-estradiol, 2.9 µg/L for 4-nonylphenol, 4.6 µg/L for 17α-estradiol and 17α-ethinylestradiol, and 8.3 µg/L for estriol. A recent study examined the determination of estrone (E1), 17β-estradiol (E2), and estriol (E3) and their conjugated metabolites levels in cow and river buffalo meat [32]. Samples were extracted with methanol, enzymatically deconjugated and purified by C18 SPE before they are analyzed by HPLC-FLD. The effect of temperature on the studied steroid concentrations was also investigated and it was shown that heating processes was not able to significantly affect the level of phenolic estrogens in meat.
Estrogens in human urine samples were determined by HPLC-FLD following vortexassisted dispersive liquid-liquid microextraction (VA-DLLME). Fine droplets of nonanoic acid floating on the top of sample solution were used to extract the estrogens using vortexmix to assist the dispersion of the extraction solvent into the aqueous sample. Derivatization was not required in this work as the high extraction efficiency of DLLME improved the sensitivity and shortened the analysis time. LOD values were 0.01 ng/ml for E3, 0.01 ng/mL for βE2, and 0.06 ng/mL for E1, respectively [82].
The abuse of anabolic steroids has gained worldwide concerns, as frequent high doses can irreversibly affect the endocrine system, mineral metabolism, and may result in hepatic carcinomas [26]. Therefore, several committees have prohibited or imposed strict rules on their use for illegal purposes, such as increasing performance activity for athletes and stimulating meat production in cattle and poultry. Amin et al. used micellar chromatography with the detergent solution as the mobile phase to detect testosterone and bolasterone in human urine samples [26]. The method made use of fluorescence by means of energy transfer from the aromatic carbonyls in the anabolic steroids to the terbium ion in micellar media. No sample preparation was required and urine samples were injected directly onto the HPLC column. Excitation of terbium by means of energy transfer from steroids resulted in 183-fold fluorescence enhancement. The detection limits were 10 ng/200 μL injection volume for testosterone and 2 ng/200 μL for bolasterone.

Detection of Endocrine Disruptive Chemicals (EDCs)
The analysis and detection of steroid blood levels is vital for the investigation of food safety and effect on health [42,83]. Many studies have reported the presence of synthetic steroids in our daily food, both from plant and animal sources [61,[84][85][86]. BPA, an industrial chemical, was found in biological fluids because of its ability to leach into food or liquids or through dental sealants into patient's saliva [32,42,[61][62][63]. One of the reasons behind their existence is that farmers increase their profit by using endocrine disruptive chemicals (EDCs) to support the feed conversion and growth rate in animals. Substances with hormonal actions are prohibited in the European community for use in animals intended for meat production due to their possible toxic effects on public health [83].
When bound to human estrogen receptors, they can stimulate the transcriptional activity of various estrogen receptor subtypes. The increase in levels of estrogen may be linked to the possibility of cancer occurrence among meat consumers [85].
In addition, studies have proved the ability of BPA to cross the placenta and blood brain barrier, in turn affecting the endocrine organs in animals and humans. This includes increasing prostate size, decreasing the number of produced sperms, and causing early A recent study examined the determination of estrone (E1), 17β-estradiol (E2), and estriol (E3) and their conjugated metabolites levels in cow and river buffalo meat [32]. Samples were extracted with methanol, enzymatically deconjugated and purified by C 18 SPE before they are analyzed by HPLC-FLD. The effect of temperature on the studied steroid concentrations was also investigated and it was shown that heating processes was not able to significantly affect the level of phenolic estrogens in meat.
Estrogens in human urine samples were determined by HPLC-FLD following vortexassisted dispersive liquid-liquid microextraction (VA-DLLME). Fine droplets of nonanoic acid floating on the top of sample solution were used to extract the estrogens using vortexmix to assist the dispersion of the extraction solvent into the aqueous sample. Derivatization was not required in this work as the high extraction efficiency of DLLME improved the sensitivity and shortened the analysis time. LOD values were 0.01 ng/ml for E3, 0.01 ng/mL for βE2, and 0.06 ng/mL for E1, respectively [82].
The abuse of anabolic steroids has gained worldwide concerns, as frequent high doses can irreversibly affect the endocrine system, mineral metabolism, and may result in hepatic carcinomas [26]. Therefore, several committees have prohibited or imposed strict rules on their use for illegal purposes, such as increasing performance activity for athletes and stimulating meat production in cattle and poultry. Amin et al. used micellar chromatography with the detergent solution as the mobile phase to detect testosterone and bolasterone in human urine samples [26]. The method made use of fluorescence by means of energy transfer from the aromatic carbonyls in the anabolic steroids to the terbium ion in micellar media. No sample preparation was required and urine samples were injected directly onto the HPLC column. Excitation of terbium by means of energy transfer from steroids resulted in 183-fold fluorescence enhancement. The detection limits were 10 ng/200 µL injection volume for testosterone and 2 ng/200 µL for bolasterone.

Detection of Endocrine Disruptive Chemicals (EDCs)
The analysis and detection of steroid blood levels is vital for the investigation of food safety and effect on health [42,83]. Many studies have reported the presence of synthetic steroids in our daily food, both from plant and animal sources [61,[84][85][86]. BPA, an industrial chemical, was found in biological fluids because of its ability to leach into food or liquids or through dental sealants into patient's saliva [32,42,[61][62][63]. One of the reasons behind their existence is that farmers increase their profit by using endocrine disruptive chemicals (EDCs) to support the feed conversion and growth rate in animals. Substances with hormonal actions are prohibited in the European community for use in animals intended for meat production due to their possible toxic effects on public health [83].
When bound to human estrogen receptors, they can stimulate the transcriptional activity of various estrogen receptor subtypes. The increase in levels of estrogen may be linked to the possibility of cancer occurrence among meat consumers [85].
In addition, studies have proved the ability of BPA to cross the placenta and blood brain barrier, in turn affecting the endocrine organs in animals and humans. This includes increasing prostate size, decreasing the number of produced sperms, and causing early puberty in females, in addition to effects on sexual differentiation [62,87]. Therefore, several HPLC-FLD methods were developed and used for quantification of EDC's.
The fluorescent reagent 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) ( Figure 15) was used by Sun et al. to detect EDC's. To the evaporated sample residue, DIB-Cl suspension in acetonitrile and triethylamine in acetonitrile were added and reacted at room temperature [63]. The study determined BPA in rat brain samples using HPLC-FLD coupled with a microdialysis [63]. A microdialysis probe was inserted into the hypothalamus of rat brains and artificial cerebrospinal fluid was used for perfusion. After the administration of a single intravenous or oral dose of BPA, concentrations were monitored in brain and plasma for 8 h. The obtained data proved that BPA could penetrate the blood brain barrier. The LOD of BPA was 0.3 ppb in 60 μL brain microdialysate at (S/N = 3).
Kuroda et al. used a similar method for determination of BPA in human blood serum and ascitic fluid samples. Samples were extracted by LLE using chloroform [62]. The LOD of BPA for both samples was 0.04 ppb at (S/N = 3). Human breast milk was also used to investigate the presence of BPA employing DIB-Cl as a fluorescent derivatizing agent [61]. Two steps of LLE were applied, and two C18 columns were used to separate DIB-BPA from the endogenous material in breast milk. The detection limit in 23 samples of healthy lactating women was 0.11 ng/mL at (S/N = 3).
The chromatographic conditions, sample preparation and detection limits of the HPLC-FLD methods that were employed in the steroid studies reviewed in this article are summarized and presented in Table 2. Table 2. HPLC-FLD method conditions used for steroid detection. The study determined BPA in rat brain samples using HPLC-FLD coupled with a microdialysis [63]. A microdialysis probe was inserted into the hypothalamus of rat brains and artificial cerebrospinal fluid was used for perfusion. After the administration of a single intravenous or oral dose of BPA, concentrations were monitored in brain and plasma for 8 h. The obtained data proved that BPA could penetrate the blood brain barrier. The LOD of BPA was 0.3 ppb in 60 µL brain microdialysate at (S/N = 3).
Kuroda et al. used a similar method for determination of BPA in human blood serum and ascitic fluid samples. Samples were extracted by LLE using chloroform [62]. The LOD of BPA for both samples was 0.04 ppb at (S/N = 3). Human breast milk was also used to investigate the presence of BPA employing DIB-Cl as a fluorescent derivatizing agent [61]. Two steps of LLE were applied, and two C 18 columns were used to separate DIB-BPA from the endogenous material in breast milk. The detection limit in 23 samples of healthy lactating women was 0.11 ng/mL at (S/N = 3).

Pharmaceutical Applications
Due to its simplicity and versatility, HPLC has become a cornerstone tool in pharmaceutical and biomedical analysis. HPLC is routinely used in drug discovery, development, and manufacturing, and routine assessment for the identification and quantification of drugs, both as active pharmaceutical ingredients and within their formulations [106]. In addition, it is essential for carrying out product characterizations, including assaying active pharmaceutical ingredients and profiling impurities [107], as well as degradation products generated by accelerated aging [108]. Moreover, the development of formulations requires studying dissolution properties, stability, and content uniformity of solid dosage forms, as well as conducting assays for the pharmaceutical formulations all of which are carried out using HPLC [106,109]. Although UV is the common detector for these applications, FLD was employed in several assays particularly in steroid analysis due to its sensitivity and selectivity as discussed below.
One area employing HPLC-FLD for steroid detection is investigating their pharmacokinetic-pharmacodynamic (PK-PD) properties. Vesser et al. [69] described HPLC-FLD method for quantification of the neurosteroids alfaxalone and pregnanolone in plasma samples ( Figure 16). The method involved sample pretreatment with acetonitrile to precipitate plasma proteins, derivatization with dansyl hydrazine, and LLE with dichloromethane. Analysis was done using isocratic reverse phase HPLC on a 3 µM Microsphere C 18 column. The limit of detection using 50 µL plasma sample was 10 ng/mL. Butane acid-(5-androsten-17-one-3ß-ol)-diester (A1998), a novel dehydroepiandrosterone (DHEA) derivative antiarrhythmic steroid was successfully quantified by HPLC-FLD. Although LC-MS has the required sensitivity for quantifying low levels of steroids, A1998 is not ionizable and hard to aerosolize and therefore was not suitable for MS detection. HPLC-FLD was therefore ideal to study its PK-PD properties in rat plasma. A mixture of ZnSO4 solution and acetonitrile were used for sample preparation to precipitate sample plasma proteins [70] and dansyl hydrazine was used as a derivatizing agent using trifluoroacetic acid (TFA) as a catalyst to enhance the yield. A C18 reverse-phase column (150 mm × 4.6 mm, 5 mm) was used for the analysis and the limit of detection was 25 ng/mL (S/N = 10) using 200 μL of plasma.
Another area employing HPLC-FLD for detection of steroids is for development and characterization of drug formulation. In a recent study HPLC-FLD was used to characterize polypeptide-corticosteroid conjugates as a topical treatment approach to treat psoriasis [110]. The study successfully synthesized and characterized a pH-responsive biodegradable poly-L-glutamic acid-fluocinolone acetonide conjugate as a controlled release treatment to reduce skin inflammation. The method employed C-18 LiChrospher analytical column (125 × 4.0 mm), with a flow rate of 1 mL/min, the using a mobile phase of H2O (1% orthophosphoric acid)/ACN (60/40) [110].
Due to the low administered dose of ethinyl estradiol, which can be as low as 10 μg/mL, methods employ derivatization or require composite of samples of up to 10 tables to improve the quantification limits in performing uniformity and dissolution tests [26,111]. Nonetheless, ethinyl estradiol is a native fluorescent, and it is therefore simpler to measure using direct methods. Strusiak   Butane acid-(5-androsten-17-one-3ß-ol)-diester (A1998), a novel dehydroepiandrosterone (DHEA) derivative antiarrhythmic steroid was successfully quantified by HPLC-FLD. Although LC-MS has the required sensitivity for quantifying low levels of steroids, A1998 is not ionizable and hard to aerosolize and therefore was not suitable for MS detection. HPLC-FLD was therefore ideal to study its PK-PD properties in rat plasma. A mixture of ZnSO 4 solution and acetonitrile were used for sample preparation to precipitate sample plasma proteins [70] and dansyl hydrazine was used as a derivatizing agent using trifluoroacetic acid (TFA) as a catalyst to enhance the yield. A C 18 reverse-phase column (150 mm × 4.6 mm, 5 mm) was used for the analysis and the limit of detection was 25 ng/mL (S/N = 10) using 200 µL of plasma.
Another area employing HPLC-FLD for detection of steroids is for development and characterization of drug formulation. In a recent study HPLC-FLD was used to characterize polypeptide-corticosteroid conjugates as a topical treatment approach to treat psoriasis [110]. The study successfully synthesized and characterized a pH-responsive biodegradable poly-L-glutamic acid-fluocinolone acetonide conjugate as a controlled release treatment to reduce skin inflammation. The method employed C-18 LiChrospher analytical column (125 × 4.0 mm), with a flow rate of 1 mL/min, the using a mobile phase of H 2 O (1% orthophosphoric acid)/ACN (60/40) [110].
Due to the low administered dose of ethinyl estradiol, which can be as low as 10 µg/mL, methods employ derivatization or require composite of samples of up to 10 tablets to improve the quantification limits in performing uniformity and dissolution tests [26,111]. Nonetheless, ethinyl estradiol is a native fluorescent, and it is therefore simpler to measure using direct methods. Strusiak et al. used reversed phase HPLC-FLD with a mobile phase of 0.05 M aqueous KH 2 PO 4 -methyl alcohol (2:3) [112] for measuring ethinyl estradiol in tablets. The method required minimal sample preparation and can be applied to measure ethinyl estradiol in combination with other compounds, such as methyl testosterone and progesterone.
Glowka et al. measured triamcinolone (TMC) in the presence of endogenous corticosteroids in human plasma, after its administration as tablets to healthy volunteers [88]. The samples were subjected to SPE before derivatization with 9-anthroyl nitrile (9-AN) in a basic mixture. An isocratic RP-HPLC was performed using a C 18 column and a mixture of acetonitrile and 0.3 mM ortho-phosphoric acid as the mobile phase. The method recorded LOD of 1.0 ng/mL (S/N = 6/1).
The use of HPLC-FLD for the analysis of commercial estrogens using post-column online photochemical derivatization was investigated by Gatti et al. [90]. Separation of conjugated and unconjugated estrogens were optimized using a Phenomenex Prodigy column 5 ODS2, and a mobile phase consisting of a TEA phosphate buffer (pH 4.0; 0.05 M) and acetonitrile at different concentrations and flow rates. The addition of photochemical derivatization to the HPLC-FLD method recorded a 1000 times higher sensitivity than UV. The LODs of conjugated estrogens were between 0.03 and 0.19 pmol (S/N = 3). This method provided useful information regarding quality of estrogen in raw materials and conjugated estrogens in dosage forms.
HPLC coupled with UV/FLD was used by Arsova-Sarafinovska et al. for determination of ethinylestradiol and levonorgestrel, present at very low levels in a low-dose oral contraceptive. The method employed Purospher ® reversed phase column (150 × 4.0 mm I.D., particle size 5 µm) and a 47%: 53% acetonitrile: water (v/v). Drospirenone, was used as internal standard. FLD showed excellent detection limit for ethinyl estradiol (0.65 ng/mL) which was 83 times lower compared to that produced by UV [12].
Similarly, Silva et al. used HPLC coupled with UV/FLD to quantify ethinyl estradiol and the synthetic progestin drospirenone in coated tablets [73]. The method conditions were comparable to that developed by Arsova-Sarafinovska et al. but reported higher detection limits using FLD (20 ng/mL) [89]. The method was fully validated and was found suitable for routine use in quality control laboratories.

Environmental and Food Applications
Steroids can be found in various environmental samples, including water, plant, and animal samples. Water is the transporter medium and reservoir for various synthetic steroids, whereas plants were found to contain many different natural steroids [17,52,[93][94][95], which can be classified according to their biological relevance. For example, plant physiological steroids can be present as hormones (e.g., brassinolide) and pheromones (e.g., antheridiol), whereas plant allelochemical substances are biologically related to animal hormones such as vertebrate hormones (progesterone) and insect hormones (ecdysone). Other plant steroids can act as protective steroids (e.g., digitoxigenin, solanidine) [51,99,113].
Exogenous estrogens can enter the human body through sewage discharge and animal waste disposal. It has been reported that the discharge of industrial and municipal wastewater can introduce up to 60% of the manufactured surfactant alkylphenol ethoxylate (APE) [42]. APE can degrade to more toxic products by sewage treatment plants (STP), such as nonylphenol (NP), octylphenol (OP), and their metabolites [28], which can cause toxicity at a low concentration range (3.7-6.0 µg/L). Sample preparation procedures and analytical methods used for analysis of steroid hormones in environmental and food samples have been recently reviewed [114].
Many studies used HPLC-FLD for the analysis of steroids in environmental samples due to its sensitivity and selectivity. Studies investigated the steroid content in samples collected from drinking water, wastewater, river water, tap surface water, and mineral and underground river water [17,20,37,52,[92][93][94][95]. Steroids were also analyzed in samples taken from effluent, influent, and sediment wastewater in treatment plant [41]. Other studies have collected samples from fish, chicken, cows, river buffalos, and measured their steroid content [31,73,95].
Lu et al. have analyzed poultry litters samples to detect estrogens 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). The samples were extracted with a mixture of dichloromethane and methanol followed by a clean-up procedure using normal-phase open column chromatography to remove lipid contents. The detection limits for E2 and EE2 were 4.0 µg/kg and 2.6 µg/kg, respectively [91].
In another study, a method was developed to detect four endocrine disruptors: 17estradiol, estriol, bisphenol A, and 17-ethinylestradiol in environmental water using online monolithic SPME with HPLC-FLD [96]. The speed of the analysis was improved using the extraction medium, poly(acrylamide-vinylpyridine-N,N methylene bisacrylamide) monolith, which was synthesized inside a polyether ether ketone (PEEK) tube. The method was applied successfully to the analysis of several types of environmental water samples. Low detection limits in the range of 0.006-0.10 ng/mL (S/N = 3) were achieved for the target compounds.
Another method based on SPME monolithic extraction media coupled with HPLC-FLD was also employed by Fan et al. to determine bisphenol A and 17α-ethinylestradiol in environmental water samples. The method achieved high extraction efficiency and produced a detection limit of 0.064 and 0.12 ng/mL for bisphenol A and 17α-ethinylestradiol, respectively. The method was applied successfully to the analysis of environmental water samples from different sources demonstrating the robustness of the method in real settings [98].
Online extraction was also employed by another group using SPE with HPLC-FLD for the quantification of selected EDCs in water. Ying et al. tested three types of SPE cartridges to preconcentrate nonylphenols, octylphenols, POE(1-2) nonyl phenol and bisphenol A as well as hormone steroids estradiol, estriol, ethinylestradiol, and ethinylestradiol 3-methyl ether in deionized water. The highest recoveries were obtained with PLRP-s and PRP-1 polymer cartridges compared to C 18 cartridges. The method was applied successfully on a river water sample spiked with the EDCs. The detection limits ranged between 20 and 50 ng/L [97].
Another recent study investigating river water samples by HPLC-FLD was carried out by Ali et al. [34]. The study employed a derivatization procedure, which targets the ethinyl group rather than the phenolic group to react with an aryl halide in the presence of palladium (Pd) and copper catalysts. The fluorescent aryl halide labeling reagent used in this reaction is 4-(4,5-diphenyl-1H-imidazol-2-yl) iodobenzene (DIB-I); was prepared by the same group. The method achieved a low detection limit (S/N = 3) of 7.4 ng/L [34].
Zhang et al. determined eight EDCs in wastewater using precolumn derivatization and SPE-HPLC-FLD. In the study 4-octylphenol, 4-nonylphenol, bisphenol A, diethylstilbestrol, estrone, 17α-ethinylestradiol, 17β-estradiol, and estriol were derivatized with 10-ethyl-acridone-2-sulfonyl chloride (EASC). The method was successfully applied to the determination of the EDC compounds in wastewater samples with a significant improvement in sensitivity compared to traditional HPLC methods. The LODs of the method were between 0.3 and 0.7 ng/L [52].
Four trace estrogens were determined in different environmental samples using solidliquid extraction/auto SPE and HPLC-FLD method developed by Liu et al. The LODs of the method were down to 1.1 × 10 −2 (estrone), 4.11 × 10 −4 (estradiol), 5.2 × 10 −3 (estriol), and 7.18 × 10 −3 µg/L (17α-ethynyl estradiol) [21]. The study that quantified estrogens in samples collected at livestock farms and a major river in Northeast China, warned that high levels were observed and that estrogenic fate and contamination should be investigated in the region [21]. The same HPLC-FLD method was employed in two recent studies investigating steroid-degrading strains of bacteria as an approach for tackling increased environmental pollution of steroids [115,116].
Municipal wastewater treatment plants were sampled to investigate the presence of the steroids nonylphenol (NP), octylphenol (OP), nonylphenol polyethoxylates (NPE), 17α-estradiol (E2), and ethinylestradiol (EE2) [5]. SPE disks were used for extraction, followed by HPLC coupled with FLD or competitive radioimmunoassay (RIA). The recorded LODs for HPLC-FLD were 11, 2, and 52 ng/L of water for NP, OP, and NPE, respectively, but were higher than those obtained with HPLC-RIA for E2 and EE2, recorded as 107 and 53 pg/L, respectively [5]. However, safety is a major limitation for routine use of RIA.
Lima et al. developed a low-cost fast method using DLLME with HPLC-FLD for quantification of estrogens in water. The method produced high recoveries and detection limits of 2.0 ng/L and 6.5 ng/L for E2 and EE2, respectively. The method that is suitable for analyzing large number of samples and uses low amount of extraction solvent was applied successfully to the analysis of tap, surface, and waste water samples [37].
The adsorption of estrogens on different types of powdered activated carbon (PAC) was investigated by Yoon et al. using HPLC-FLD. The study demonstrated the feasibility of using PAC for removal of >99% of BPA, 17 β-estradiol (E2), and 17α-ethynyl estradiol (EE2) from raw drinking waters at least at initial concentration of 500 ng/L and higher [94]. PAC type, dosage, and the presence or absence of natural organic matter determined the percentage of steroid removal. The removal was improved by increasing both PAC dose and contact time. The HPLC-FLD method had detection limits of 0.88, 1.15, and 0.96 nM for BPA, E2, and EE2, respectively.
Kozłowska-Tylingo et al. compared the different methods and extraction procedures for the analysis of five estrogens in drinking water and wastewater samples. Extraction of 17α-ethinylestradiol, estrone, estradiol, estriol, and progesterone produced the best recoveries using extraction disks compared to C 18 extraction columns. SPE-LC-MS/MS produced the best sensitivities compared to other methods employing UV and FLD [19].
Zhang et al. investigated ionic liquid based pretreatment for quantification of three steroids in water samples by HPLC-FLD. The sample pretreatment has the advantages of being safer and environmentally friendly since no organic solvents is used. The method based on ionic liquid-based homogeneous liquid-liquid microextraction (IF-IHLME) produced detection limits of 0.04, 0.05, and 0.05 ng/mL for 17-α-estradiol, 17-β-estradiol-benzoate, and quinestrol, respectively [101].
Estrogens and their metabolites were also quantified in a study by Guedes-Alonso et al. using molecularly imprinted (MIP) solid-phase extraction coupled with UPLC-FLD. The method was used to analyze wastewater from a veterinary hospital as well as influent and effluent samples of a wastewater treatment plant. The limits of detection were between 0.18 and 0.45 ng/mL [41].
The occurrence of trenbolone acetate metabolites, which is widely used to promote the growth of beef cattle, was evaluated in samples from a beef cattle feedlot discharge and in river water upstream and downstream from the discharge [95]. HPLC-FLD using methanol and water as the mobile phase in gradient flow and C 18 column with dimensions of 4.6 mm × 250 mm was employed to quantify 17α-and 17β-trenbolone in the samples. The recorded LOD for was around 4 ng/L. The same metabolites were also quantified in bovine muscle, and liver samples by SPE-HPLC-FLD [33]. In the study, Yoshioka et al. examined several extraction solvents, such as methanol, diethyl ether, acetonitrile, and ethyl acetate, with the latter resulting in the cleanest extracts [33]. LODs in liver and bovine muscle samples were 0.2 and 1.0 ng/g, respectively.
Determination of natural and synthetic estrogenic compounds in dairy products was carried out using hollow fiber liquid-phase microextraction coupled to HPLC-FLD/PDA. Estriol, 17β-estradiol, 17α-estradiol, estrone, 17α-ethinylestradiol, diethylstilbestrol, dienestrol, and hexestrol) and 2-hydroxyestradiol were quantified in natural yogurt, a probiotic yogurt-type drink and cheese. The method produced LODs in the low lg/kg or lg/L range with good precision and accuracy [36].
Ultrasonic assisted dispersive liquid-liquid microextraction (UA-DLLME) method coupled with 9-phenanthreneboronic acid derivatization was used for the quantification of brassinolide a plant hormone by HPLC-FLD. Different extraction and degravitation conditions were investigated and optimized. The method was applied for brassinolide determination in Arabidopsis thaliana, Daucus carota and Brassica campestris L. leaves with higher sensitivity than similar reported methods. LOD of the method was 8.0 ng/L [99].
A study by Ito et al. used HPLC-FLD method for the quantification of different phytosterols and cholesterol in land plants and marine algae after derivatization with 1-anthroyl nitrile. The method used a C-30 column and an isocratic mobile phase of acetone/acetonitrile/hexane/water (71:20:4:5, v/v) at 1.0 mL min −1 . The LODs for the different sterols were between 0.25 and 0.40 µg/mL [117].
Zearalenone an estrogenic mycotoxin that contaminates cereal crops was quantified in edible oil by an automated SPE-HPLC-FLD. Dryzmala et al. employed a SPE for reversible hydrazone formation by zearalenone and a hydrazine moiety covalently attached to a solid phase. The results of the HPLC-FLD method were found in agreement with an isotopic dilution LC-MS/MS method. The LOD of the method was 10 µg/kg [100].
Alkylphenol polyethoxylates are nonionic surfactants that act as EDCs and can exert estrogenic effects on fish [118,119]. To assess their contamination in fish and shellfish samples, Tsuda et al. developed a simple and highly sensitive HPLC method [32]. The method was used to detect 4-nonylphenol (NP), 4-nonylphenol mono-(NP1EO), diethoxylates (NP2EO), bisphenol A (BPA), 4-tert-butylphenol (BP), and 4-tert-octylphenol (OP). Acetonitrile was used for extraction, followed by clean-up with a magnesia silica gel adsorbent, Florisil PR, before analysis with HPLC-FLD. The mobile phase was water and methanol with gradient flow. The detection limit was 2 ng/g for NP, NP1EO, and NP2EO, and 1 ng/g for BPA, BP, and OP.
Similarly, Datta et al. examined fish tissue for presence of alkylphenolic compounds using an HPLC-FLD method [28]. Extraction of ground fish tissue was conducted using pressurized fluid extraction, followed by purification on amino propyl silica cartridges. Hexane and ethanol were used as the mobile phase. The recorded LOD was 4-15 ng/mL.

Conclusions
This review summarizes the various HPLC-FLD methods reported on in the literature for the quantification of steroids in clinical, pharmaceutical, food, and environmental samples. Researchers in different fields employed HPLC-FLD to meet the needs of their applications in terms of sensitivity and selectivity. Since most steroids lack inherited fluorescent properties, various fluorophore labels were introduced to target steroids through different derivatization approaches to enhance steroids quantification. Reaction schemes included in this review illustrated the chemical coupling of the fluorescent label to steroid compounds and highlighted how different steroids can be selectivity targeted depending on the functional group present in the steroid. It was shown that derivatization approaches vary according to speed, sensitivity, steroid type, the need for sample preparation/purification, and the functional group targeted, therefore offering researchers a selection of options that suit their applications. For this purpose, a table that summarizes the various derivatization approaches used and specific conditions reported on is included in this review, to help researchers quickly compare between these approaches. Another table focuses on the chromatographic and analytical method conditions, highlighting performance characteristics, such as LOD and LLOQ, and a sample matrix, which are critical for choosing the most relevant method, depending on the steroid levels and sample type. Most of the HPLC-FLD methods reported were applied in the clinical field and its related research, which is expected, due to the need for higher selectivity and sensitivity that is relevant to the complex nature of the matrix and low steroid levels of the samples. HPLC-FLD methods also found applications in the pharmaceutical and environmental fields, where sample preparation was mainly employed to enrich the low levels of steroids found in less complex sample matrices, such as water. Overall, this review provides a comprehensive summary of the various methods and procedures which are reported in the literature that can be applied to quantify steroids by HPLC-FLD.