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Pyrene Sulphonyl Chloride as a Reagent for Quantitation of Oestrogens in Human Serum Using HPLC with Conventional and Laser‐Induced Fluorescence Detection
Author(s) -
DeSilva Kapila H.,
Vest Floyd B.,
Karnes H. Thomas
Publication year - 1996
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/(sici)1099-0801(199611)10:6<318::aid-bmc604>3.0.co;2-y
Subject(s) - chemistry , detection limit , derivatization , chromatography , reagent , bromide , dansyl chloride , fluorescence , high performance liquid chromatography , chloride , analytical chemistry (journal) , organic chemistry , physics , quantum mechanics
A highly sensitive HPLC method with conventional and laser‐induced fluorescence detection for the analyses of oestrogens is described. β‐oestradiol (β‐ES) was chosen as a model and derivatized with pyrenesulphonyl chloride (PSCL), a novel derivatizing reagent, using a two‐phase system with tetrapentylammonium bromide (TPABR) as a phase transfer catalyst. Derivatization was complete in 30 s at room temperature and concentrations as low as 5×10 −10 M could be derivatized and detected. The concentration detection limit of PSCL derivatized β‐ES was 2×10 −11 M which corresponds to an on‐column detection limit of one femtomole using conventional chromatography and detection. The relative standard deviation ( n =6) of the derivatization carried out at 2.5×10 −9 M was 4%. No significant loss of peak‐height of the derivative was observed at room temperature over a 24 h period. Baseline resolution of PSCL derivatized oestrone, equilin and β‐estradiol was achieved with ACN:H 2 O (75:25 v/v) using a Bondclone C‐18 column. The method described here is sufficiently sensitive for analyses of β‐estradiol at low pg/mL concentrations in 1 mL of human serum. The PSCL derivatives of oestrogens demonstate excellent potential for excitation by laser‐induced fluorescence using the 351 nm line of an Ar ion laser or the 325 nm line of He‐Cd laser. A concentration limit of detection of 4×10 −12 M for β‐oestradiol was achieved using the 325 nm line of an He–Cd laser. This corresponds to 0.2 fmol of derivatized β‐oestradiol on‐column.