Nanoelectrospray mass spectrometry and precursor ion monitoring for quantitative steroid analysis and attomole sensitivity

Nanoelectrospray ionization (nanoESI) mass spectrometry was performed on na turally occurring steriod sulfates and unconjugated steroids derivatized to their sulfate esters using precursor ion monitoring. Initially, an ex-trac tion method was developed based on a combinatorial approach employed to obt ain the most efficient liquid/liquid extraction protocol. The new method al lowed unconjugated steroids and their sulfated analogues to be isolated sep arately in a two-step procedure using diethyl ether/hexane (90:10, v/v) in the first step to extract the unconjugated steroids and chloroform/2-butano l (50:50, v/v) in the second step to extract steroid sulfates. Precursor io n scanning performed with a triple-quadrupole mass spectrometer was used to examine quantitatively the extracted unconjugated and sulfated steroids, w here the recovery efficiency averaged 70 and 87%, respectively, In addition , some steroids could be structurally elucidated by employing tandem mass s pectrometry. The limit of detection for steroid sulfates from the biologica l matrix was 200 amol/mu L (similar to 80 fg/mu L) with only 1 mu L of samp le being injected. Endogenous levels of the unconjugated and sulfated stero ids were detected and quantified from physiological samples including urine and blood. Internal standards, pregnenolone-d(4) sulfate and dehydroepiand rosterone-d(2) (DHEA), were used for quantitation, Extraction and nanoESI a nalyses were also performed on cerebrospinal fluid where the neurosteroid D HEA sulfate was detected. The small amount of material consumed (typically less than 20% of the injection volume) suggests that nanoESI has even great er potential for high sensitivity when combined with nanoLC approaches, esp ecially for monitoring reproductive and adrenal steroids, as well as for th e analysis of the less abundant neurosteroids.