Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison.
Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.
Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Helsinki, Viikinkaari 5E, FIN-00014 Helsinki, Finland.
Laura
L
Hintikka
Tiia
T
Kuuranne
Antti
A
Leinonen
Mario
M
Thevis
Wilhelm
W
Schänzer
John
J
Halket
David
D
Cowan
Joachim
J
Grosse
Peter
P
Hemmersbach
Michel W F
MW
Nielen
Risto
R
Kostiainen
D045930:Anabolic Agents / Q000737:chemistry / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D004300:Doping in Sports; D020719:Glucuronides / Q000737:chemistry / Q000652:urine*; D006801:Humans; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D013256:Steroids / Q000737:chemistry / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry
D045930:Anabolic Agents; D020719:Glucuronides; D013256:Steroids