Gas chromatographic determination of catecholestrogens following isolation by solid-phase extraction.
A sensitive and specific assay for the determination of the catecholestrogens 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) using gas chromatography with electron-capture detection (GC-ECD) is described. The formation of 2- and 4-OHE2 was assessed following activation of 17beta-estradiol in the microsomal fraction of female rat livers. The analytes were isolated by solid-phase extraction, derivatized to their heptafluorobutyryl esters with heptafluorobutyric acid anhydride, and subjected to solvent exchange prior to analysis; this resulted in minimal chromatographic interference, long column life, and stable derivatized analytes. Derivatized catechols were separated and confirmed with dual column chromatography (DB-5 and DB-608) and quantitated using GC-ECD. The DB-608 column was preferred for quantitation as it provided better 4-OHE2 resolution from interference. Key validation parameters for the assay include sensitivity, intra- and inter-assay precision, and accuracy. Instrument sensitivity and limits of detection (LOD) and quantitation (LOQ) were determined statistically from fortification data approaching expected limits. For 2-OHE2 and 4-OHE2, respective values for these parameters were; instrument sensitivities of 0.4 and 0.7 pg, LODs of 0.8 and 1.3 ng/mg, and LOQs of 2.6 and 4.3 ng/mg.
Pinnella
K D
KD
Department of Environmental Health, Colorado State University, Fort Collins 80523, USA.
Cranmer
B K
BK
Tessari
J D
JD
Cosma
G N
GN
Veeramachaneni
D N
DN
D000818:Animals; D002849:Chromatography, Gas / Q000379:methods*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000032:analysis*; D002393:Estrogens, Catechol; D005260:Female; D008862:Microsomes, Liver / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002393:Estrogens, Catechol; D004958:Estradiol; C001390:2-hydroxyestradiol; C014036:4-hydroxyestradiol