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 high performance liquid chromatography (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.
|Reagent||Target Steroid||Sample Preparation and Derivatization Conditions||Reference|
||Estrogens||Sample was extracted by SPE C18.
Residue reacted with p-nitrobenzoyl chloride at 25 °C, 30 min
|Mao et al. 2004 |
|1-Anthroyl nitrile (1-AN)
||18-Oxygenated corticosteroids, 18-hydroxycortisol, 18-hydroxycortisone and 18-oxocortisol Pregnenolone and C21 steroids; especially corticoids||Sample was extracted by SPE or LLE with a mixture of diethyl ether and dichloromethane.
Extract reacted with 1-anthroyl nitrile in acetonitrile at room temperature for 10 min.
|Kurosawa et al. 1995 
Shimada et al. 1996 
Shimada et al. 1991 
|9-Anthroyl nitrile (9-AN)
F and E, Hydroxysteroids
|Sample was extracted by LLE or SPE.
Extracts reacted with 9-AN in a mixture of quinuclidine and triethylamine for 30 min at RT.
|Glowka et al. 2009 
Goto et al. 1983 
Shibata et al. 1997 
Haegele et al. 1991 
Kosicka et al. 2018 
Neufeld et al. 1998 
Shimada et al. 1991 
|2-(11H-Benzo[a]carbazole-11-yl) ethyl carbonochloridate (BCEC-Cl)
||E1, E2, E3, BPA, NP, OP||Sample was extracted by DLLME.
Extracts were added to BCEC-Cl in a NaHCO3 buffer at pH 10; it was shaken for 10 s, and then allowed to stand for 14 min at 43 °C.
|Wu et al. 2015 |
|Brassinosteroids||Sample was extracted by LLE.
Extracts reacted with 9-phenanthrene-boronic acid in a mixture of pyridine and acetonitrile for 10 min at 70 °C.
Takatsuto. 1989 
|Sample was extracted by LLE or SPE.
Extracts reacted with EASC in anhydrous acetonitrile and NaHCO3 buffer pH (10.2), for 5 min at 60 °C.
|Zhang et al. 2012 |
|Sample was extracted by SPE or LLE.
Extracts reacted with sulfuric acid in ethanol; it was cooled in crushed ice for 15 min in the dark.
|Nozaki et al. 1991 
Nozaki et al. 1992 
Sudo et al. 1990 Gao et al. 2010 
|7-Methoxycoumarin-3-carbonyl azide (MC-CON3)
||7α-hydroxycholesterol||Sample was extracted by LLE and purified by normal-phase SPE.
Extracts reacted with MC-CON3 in ethyl acetate (AcOEt) for 40 min at 140 °C.
|Saisho et al. 1998 |
||Corticosteroids, BPA and alkylphenols||Sample was extracted by LLE.
0.02% w/v CDB solution, 2.0% w/v 1-isopropyl-3-(3-dimethylaminopropyl) carbodiimide perchlorate (IDC) solution, and 0.01% w/v (10 mg/mL) 4-piperidinopyridine, for 60 min at 40 °C.
|Katayama et al. 1991 
Katayama et al. 1992 
Katayama et al. 2001 
||17-hydroxycorticosteroids||Sample was extracted by LLE.
Extracts reacted with benzamidine in a basic medium of sodium hydroxide solution, with a mixture of propanol and water for 5 min at 95 °C.
|Seki et al. 1984 |
imidazol-2-yl) benzoyl chloride (DIB-Cl)
|BPA||Sample was extracted by LLE or SPE.
Extracts reacted with DIB-Cl in a mixture of acetonitrile and triethylamine for 20 min at RT.
|Sun et al. 2004 
Kuroda et al. 2003 
Sun et al. 2002 
|4-(4,5-Diphenyl-1H-imidazol-2-yl) iodobenzene (DIB-I)
||EE||Sample was extracted by the SPE disc method (C18 SPE disk).
A total of 50 μL of the extracts reacted with 50 μL of DIB-I (3.0 mM), in a mixture of 50 μL of solution containing 0.2 mM of PdCl2 and 0.3 mM of CuI, and 50 μL of DIPEA (3.0 mM). The vial contents were deoxygenated by N2 purge for 10 sec, heated at 100 °C for 40 min, cooled, then filtered through a 0.45-μm membrane filter before injection into the HPLC-FLD system.
|Ali et al. 2020 |
||α-Dicarbonyl compounds; prednisolone, PN and 21-hydroxycorticosteroids||Sample was extracted by LLE.
Extracts reacted with DMB for 40 min at 60 °C.
|Yamaguchi et al. 1991 
Yoshitake et al. 1989 
Yamaguchi et al. 1989 
|2-(4-Carboxyphenyl)-5,6-dimethylbenzimidazole (BODIPY FL hydrazide)
||Aldehydes and ketones; progesterone,
17-hydroxyprogesterone, and other 3-keto steroids.
|Sample was extracted by LLE.
Extracts reacted with BODIPY FL hydrazide in ethanol for 15 h at RT.
|Katayama et al. 1998 |
||F, Butane acid-(5-androsten-17-one-3beta-ol)-diester (A1998), alfaxalone and pregnanolone ||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 
Visser et al. 2000 
Peng et al. 2007 
|Naproxen acyl chloride in toluene
||Cholesterol and sitosterol||Sample was extracted by LLE.
Extracts reacted with naproxen acyl chloride in toluene, while shaking for 1.5 h at 90 °C. Diethylamine in toluene was then added to inactivate the excess naproxen acyl chloride, while shaking for 5 min at 30 °C.
|Lin et al. 2007 |
||Brassinolide and castasterone||Sample was extracted by LLE.
Extracts reacted with dansylaminophenylboronic acid in a mixture of pyridine and acetonitrile for 20 min at 70 °C.
|Motegi et al. 1994 |
|9-Fluorenylmethyl chloroformate (Fmoc-Cl)
||E1, E2, E3, BPA, NP, OP||Samples were extracted by MSPE.
Extracts were reacted with Fmoc-Cl in NaHCO3 (pH = 10.5) at 60 °C for 10 min, and then added to a mixture of aqueous acetic acid and acetonitrile. The mixture was cooled to RT.
|Qianyu Li et al. 2018 |
|FFA||The samples were extracted by supercritical CO2 and organic solvent extraction. The extracted samples were derivatized in BCETS in a solution of K2CO3 at 84 °C; then the mixture was cooled down to RT and diluted with DMF.||Li et al. 2011 |
|Benzimidazo[2, 1-b]quinazoline-12(6H)-one-5-ethylimidazole ester (BQEIC)
||OP, NP, TBP, BPA, E1, E2, E3||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.
|Liu et al. 2018 |