Liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma.
BACKGROUND
Estradiol (E2) and estrone (E1) measurements form an integral part of the assessment of female reproductive function and have expanding roles in other fields. However, many E1 and E2 immunoassays have limited functional sensitivity, suffer from cross-reactivity, and display poor intermethod agreement. To overcome these problems, we developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of E1 and E2.


METHODS
After dansyl chloride derivatization, samples were separated by fast gradient chromatography and injected into a tandem mass spectrometer after formation of positive ions with atmospheric pressure chemical ionization. The limits of detection and quantification, recovery, linearity, precision, and reference intervals were determined, and performance was compared with several immunoassays.


RESULTS
Total run time per sample was 5 min. The multiple-reaction monitoring ion pairs were m/z 506/171 for 3-dansyl-estradiol and m/z 504/171 for 3-dansyl-estrone. The limits of detection for E1 and E2 were 12.9 pmol/L (3.5 ng/L) and 10.3 pmol/L (2.8 ng/L), respectively. Interassay imprecision (CV) was 4-20% (n = 20). The limits of quantification (functional sensitivities) for E1 and E2 were 44.1 pmol/L (11.9 ng/L) and 23.2 pmol/L (6.3 ng/L), respectively. The assay was linear to >2200 pmol/L ( approximately 600 ng/L) for each analyte. Recoveries were 93-108% for E1 and 100-110% for E2. No cross-reactivity was observed. Method comparison with several immunoassays revealed that the latter were inaccurate and prone to interferences at low E1 and E2 concentrations.


CONCLUSIONS
LC-MS/MS allows rapid, simultaneous, sensitive, and accurate quantification of E1 and E2 in human serum.
Nelson
Robert E
RE
Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Grebe
Stefan K
SK
OKane
Dennis J
DJ
Singh
Ravinder J
RJ
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002853:Chromatography, Liquid; D004958:Estradiol / Q000097:blood*; D004970:Estrone / Q000097:blood*; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D010949:Plasma; D012016:Reference Values
D004970:Estrone; D004958:Estradiol