Multi residue method using coupled-column HPLC and GC-MS for the determination of anabolic compounds in samples of urine
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Series / Report no.
Open Access
Type
Report
Language
en
Date
1998-11-01
Research Projects
Organizational Units
Journal Issue
Title
Multi residue method using coupled-column HPLC and
GC-MS for the determination of anabolic compounds in samples of
urine
Translated Title
Multi-residu methode voor anabolica in
urinemonsters met kolomschakeling HPLC en GC-MS
Published in
Abstract
Dit rapport beschrijft een multiresidu methode voor de
analyse, detectie en identificatie van residuen van anabolica. De
analytische methode is gebaseerd op een geautomatiseerde vaste faseextractie
en een High Performance Liquid Chromatography (HPLC)
kolomschakelingssysteem. Detectie vindt plaats met Gas Chromatografie met
Massa Spectroscopische Detectie (GC-MS). De methode is geschikt voor de
screening van urine monsters op 17 anabolica en de metabolieten daarvan.
Gedeutereerde analoga worden gebruikt als interne standaarden voor
kwantificering en kwaliteitscontrole. Na screening kan de identiteit van de
anabolica bevestigd worden door het gebruik van een gemodificeerde
HPLC-procedure met gradintelutie en selectieve fractionering. De
geautomatiseerde extractie wordt uitgevoerd door een Gilson ASPEC systeem
met wegwerp vaste fase extractie kolommen gekoppeld aan een HPLC met
kolomschakeling. Het kolom schakelsysteem bestaat uit twee analytische
kolommen gekoppeld via een schakelklep. Na extractie en HPLC zuivering
wordt voor de screening een enkele fractie uitgevangen die alle analieten
bevat. Deze fractie wordt in tweeen gedeeld en gederivatiseerd met twee
verschillende derivatiseringsreagentia voor analyse met GC-MS. De
detectielimiet voor alle componenten, behalve diethylstilbestrol (DES), is 1
microg/l. Voor DES bedraagt deze limiet 0.1 microg/l. De meerderheid van de
componenten heeft een nauwkeurigheid tussen de 90 en 110 %. Voor drie
componenten (4-chloro-4-androst-3,17-dione, methyltestosterone en
17-beta-nortestosterone) werd een grotere spreiding waargenomen. Voor alle
componenten is de nauwkeurigheid acceptabel. Deze screeningsmethode is
getest op vals negatieve resultaten door het analyseren van runder- (n=20),
schapen- (n=20) en varkens- (n=10) urines op het concentratie niveau van n
en twee maal de detectielimiet. De resultaten werden acceptabel geacht als
het aantal vals negatieve resultaten kleiner of gelijk was aan 5%. Voor
runder- en varkensurines op het niveau van 1 microg/l werd aan deze eis
voldaan. Voor monsters schapenurines werden de resultaten op het
concentratie van 2 microg/l geaccepteerd. De methode kan ook gebruikt
worden als bevestigingsmethode. Voor dit doel wordt de te bevestigen
analiet selectief gesoleerd door HPLC met gradientelutie en fractionering.
De uitgevangen fractie wordt gederivatiseerd en bevestigd met GC-MS door het
meten van ten minste 4 specifieke massafragmenten. De analysemethode is in
detail beschreven in ARO SOP 401
This report describes a multi-residue method for the detection and identification of residues of anabolic compounds. The method is based on automated sample preparation (Solid Phase Extraction (SPE)) and coupled-column High Performance Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS). The method is suitable for the screening of samples of urine for the presence of 17 anabolic compounds or their metabolites. For purposes of quantification and quality control (QC) a number of deuterated internal standards is used. Following screening the identity of any of these compounds can be confirmed using a modified HPLC procedure with gradient elution and selective fractionation. The extraction of samples of urine is performed automatically with a Gilson Aspec System equipped with disposable SPE columns in combination with coupled column reversed phase HPLC. The coupled-column system consists of two analytical columns coupled via a switching valve. After extraction and HPLC clean-up a single fraction containing all the analytes is collected. This fraction is divided into two parts for derivatization using two different procedures; final determination is done with GC-MS. The limit of detection for all compounds, except diethylstilbestrol (DES), is 1 microg/l (ppb). For DES this limit is 0.1 ppb. For the majority of compounds the accuracy is between 90% and 110%. For only three compounds (CLAD, MT and 17beta-NT) were slightly larger deviations observed. In all cases the accuracy is considered acceptable. The procedure for screening was tested for false negative results by spiking samples of bovine- (n=20), ovine- (n=20) and porcine urine (n=10) both at the level of the detection limit and two times this level. The results were considered "acceptable" if the number of false negative results was equal to or below 5%. The results for samples of bovine and porcine urine were in most cases acceptable at the level of 1 microg/l. For samples of ovine urine the results were acceptable at the level of 2 microg/l. The fraction obtained when analysing the target analyte selectively using HPLC with gradient elution is suitable for confirmation of the analyte based on the detection of at least four ions. The procedure is described in detail in SOP ARO/401.
This report describes a multi-residue method for the detection and identification of residues of anabolic compounds. The method is based on automated sample preparation (Solid Phase Extraction (SPE)) and coupled-column High Performance Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS). The method is suitable for the screening of samples of urine for the presence of 17 anabolic compounds or their metabolites. For purposes of quantification and quality control (QC) a number of deuterated internal standards is used. Following screening the identity of any of these compounds can be confirmed using a modified HPLC procedure with gradient elution and selective fractionation. The extraction of samples of urine is performed automatically with a Gilson Aspec System equipped with disposable SPE columns in combination with coupled column reversed phase HPLC. The coupled-column system consists of two analytical columns coupled via a switching valve. After extraction and HPLC clean-up a single fraction containing all the analytes is collected. This fraction is divided into two parts for derivatization using two different procedures; final determination is done with GC-MS. The limit of detection for all compounds, except diethylstilbestrol (DES), is 1 microg/l (ppb). For DES this limit is 0.1 ppb. For the majority of compounds the accuracy is between 90% and 110%. For only three compounds (CLAD, MT and 17beta-NT) were slightly larger deviations observed. In all cases the accuracy is considered acceptable. The procedure for screening was tested for false negative results by spiking samples of bovine- (n=20), ovine- (n=20) and porcine urine (n=10) both at the level of the detection limit and two times this level. The results were considered "acceptable" if the number of false negative results was equal to or below 5%. The results for samples of bovine and porcine urine were in most cases acceptable at the level of 1 microg/l. For samples of ovine urine the results were acceptable at the level of 2 microg/l. The fraction obtained when analysing the target analyte selectively using HPLC with gradient elution is suitable for confirmation of the analyte based on the detection of at least four ions. The procedure is described in detail in SOP ARO/401.
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