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ChromJournalInnoVAtIVe pRoducts foR chRomAtoGRAphyAnalysis of testosterones usinga core enhanced technologyAccucore hplc columnJoanne Gartland, Thermo Fisher Scientific, Runcorn, Cheshire, UKThis demonstrates theuse of the ThermoScientific Accucore RP-MS HPLC column forthe fast analysis of threetestosterones.Introductionaccucore HPlC columns use Core enhancedTechnology to facilitate fast and high efficiencyseparations. The 2.6 μm diameter particles arenot totally porous, but rather have a solid coreand a porous outer layer. the optimised phasebonding creates a series of high coverage,robust phases. accucore RP-MS uses anoptimised alkyl chain length for more effectivecoverage of the silica surface. this coverageresults in a significant reduction in secondaryinteractions and as a result highly efficient peakswith very low tailing. the tightly controlled 2.6μm diameter of Accucore particles providesmuch lower backpressures than typically seenwith sub 2 μm materials. 11-ketotestosterone,19-nortestosterone and epitestosterone are allrelated to testosterone. 11-ketotestosteroneis an oxidised form of testosterone.19-nortestosterone contains an additionalmethyl group in comparison to testosteroneand epitestosterone and is an inactive epimer oftestosterone. all three compounds are anabolicsteroids, which promote protein synthesis withincells, particularly in muscles. 19-nortestosteronefigure 1.Chromatogram for 11-ketotestosterone (1), 19-nortestosterone (2) andepitestosterone (3) separated on an accucore RP-MS 2.6 μm100 × 2.1 mm columnoccurs naturally at low levels (0.4 ng/ml) in thebody and is routinely tested for in athletes.the separation of these three compounds andmore significantly the speed of analysis aredemonstrated in this application.experimental conditionssample preparationWorking standard contained 50 μg/ml of eachtestosterone in mobile phasecolumnaccucore RP-MS 2.6 μm, 100 × 2.1 mm (Cat.no. 554-1885)Measured pressure: 300 barAccela hplc systemColumn temperature: 40 °CInjection volume: 1 μlFlow rate: 0.6 ml/minUV detection: 54 nmmobile phase60:40 (v/v) water/acetonitrileconsumablesHPlC grade waterHPlC grade acetonitrileNSC Mass Spec Certified 2 ml clear vial withblue bonded PtFe silicone capResultsthe analysis was carried out on an accucore RP-MS 2.6 μm, 100 × 2.1 mm column.11-ketotestosterone, 19-nortestosterone andepitestosterone eluted in under 2 minutes(Figure 1).Replicated injections of the testosterone mixshowed that accucore RP-MS produced stableand reproducible results (table 1).table 1. Method precision (%RSD) for 11-ketotestosterone, 19-nortestosteroneand epitestosterone (data calculated from six replicate injections)Peak 111-ketotestosteronePeak 219-nortestosteroneAsymmetry 1.48 1.28 1.11%RSD Tr 0 0.05 0.06%RSD peak area 0.31 0.26 0.44Peak 3epitestosteroneconclusionsaccucore RP-MS columns successfullyseparated 11-ketotestosterone,19-nortestosterone and epitestosterone in lessthan 2 minutes, providing an excellent choice forthe fast analysis of testosterones, allowing highsample throughput.DescriptionCat. No.Accucore RP-MS 2.6 μm, 100×2.1 mm 554-188510VWR InteRnatIonal I chromJournal Issue 12 I 2012