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©2013 Waters Corporation INTRODUCTION Estrogen metabolites are implicated in various disease states including cancer. Clinical research into disease mechanisms related to urinary estrogen depends on the simultaneous quantitative analysis of multiple metabolites. Conventional gas or liquid chromatography in combination with mass spectrometry are commonly used for this analysis. Higher sample through-put to generate larger statistically significant data sets of estrogen compounds would be desirable. The similar, sometimes isobaric, structures of estrogen metabolites require a reliable and unambiguous chromatographic separation for reliable measurement. Analysis of Estrogen Metabolites by UltraPerformance Convergence Chromatography ® Kenneth W Blakeslee Jr, Thomas DePhillipo 1 Waters Corporation, Milford, MA USA References (1) Analysis of Fifteen Estrogen Metabolites Using Packed Column Supercritical Fluid Chromatography−Mass Spectrometry Xia Xu, Anal. Chem., 2006, 78, 1553-1558 (2) Rapid Analysis of Endogenous Steroids by Using UPC 2 MS/MS for Clinical Research Christopher J. Hudalla 1 , Stuart Chadwick 2 , Fiona Liddicoat 2 , Andrew Peck 1 and Kenneth J. Fountain 1 1 Waters Corporation, Milford, MA USA, 2 Waters Corporation, Manchester, UK CONCLUSIONS UltraPerformance Convergence Chromatography provides a rapid four minute separation of five key estrogen metabolites, including three isobaric species. The efficiency of UPC 2 creates a separation which is faster than conventional supercritical liquid chromatography or high performance liquid chromatography. The sensitivity of this method using UPC 2 and tandem mass spectrometry is in the range of sub-picograms injected on column for neat estrogen standards derivatized with dansyl chloride. The ability of this method to quantitate an expanded panel of metabolites in urine must be explored to determine it's potential as a tool for clinical research and diagnostics. Figure 1 Eliassen A H et al. Cancer Res 2012;72:696- 706 SUPERCRITICAL FLUID CHROMATOGRAPHY A study by Xia Xu (1) highlighted the ability of Supercritical Fluid Chromatography-Mass Spectrometry to generate fast separations of significant estrogen metabolites derivatized with dansyl chloride. Importantly for mass spectrometry analysis, isobaric species were separated, albeit with the use of two different SFC columns plumbed in series. The analysis speed by SFC was 7X faster than HPLC, reducing the run time from 70 minutes to 10 minutes. CONVERGENCE CHROMATOGRAPHY The present study utilizes a new form of super critical liquid chromatography, UltraPerformance Convergence Chromatography or UPC 2 , to separate estrogen metabolites. UPC 2 combines sub-2 micron particles, an optimized instrument and the ultra-low viscosity of supercritical carbon dioxide to exceed even the efficiency of classic UPLC, demonstrated by the van Deemter plots in Figure 2. A rapid and sensitive analysis by UPC 2 and tandem mass spectrometry of a mixture of underivatized estrogen and other steroids in human plasma has been previously demonstrated (2). The goal of this study was to determine if the increased efficiency of UPC 2 would separate the isobaric metabolites 16Epiestriol, 17Epiestriol, and Estriol (E3) using only one column chemistry and also provide a faster analysis than either conventional supercritical liquid chromatography or high performance liquid chromatography. METHODS INITIAL METHOD DEVELOPMENT Column Screening: A mixture containing 50 ng/mL each of Estrone (E1), Estrodiol (E2), 16Epiestriol, 17Epiestriol, and Estriol (E3) was derivatized with dansyl chloride following published protocols 1 . The final sample diluent was 100ul of 0.1M sodium bicarbonate and 100 uL of 0.1 mg/mL dansyl chloride in acetone. This mixture was used to screen two columns with a generic gradient to determine which would provide the best separation. Columns: ACQUITY UPC 2 BEH 2-Ethylpyridine 2.1x50mm 1.7um ACQUITY UPC 2 BEH Amide 2.1x50 1.7um Screening Conditions: Instrument: ACQUITY UPC 2 Xevo TQ-S MS 515 makeup pump Mobile Phase A: Liquid CO 2 (beverage grade) Modifier B: Methanol Needle Wash: 70/30 MeOH/IPA Flow Rate: 2.5 mL/min Gradient: 5% to 25% Modifier B in 3 minutes. equilibration at 5% modifier B for 1 min. Column Temp: 60°C CCM Pressure: 1800 psi Data System MassLynx 4.1 MakeUp Flow: 200uL/min Mass Spec Method Transitions were optimized by direct infusion of standards into the Xevo TQ-S using the on-board fluidics, without connection to the UPC2. Using the Xevo IntelliStart function, determination of optimal transitions in electrospray positive mode was made. The mass spec system was then coupled to the UPC2 system using a Mass Spec Splitter, incorporating the addition of a make-up flow pump to facilitate sample flow into the MS and subsequent ionization (Figure 3). A flow splitter was employed to divert a portion of the mobile phase to the mass spec and simultaneously maintain required pressure on the CO2. A makeup flow at 200uL/min of 0.1% formic acid in methanol was maintained to enhance analyte ionization. Name Mode MRM Cone Voltage Dwell Time Collisio n Energy E1 ES+ 504.29>171.16 45 0.247 30 E2 ES+ 506.26>171.16 45 0.247 30 E3, 16epi, 17epi ES+ 521.90>171.16 45 0.247 30 Table 1 Transitions determined by IntelliStart. Fragment 171.16 is a common fragment for estrogen metabolites derivatized with dansyl chloride. Sample Prep 200uL of Estrogen metabolites dissolved in methanol with 0.1% ascorbic acid were dried under nitrogen. To the dried samples were added 100uL of sodium bicarbonate buffer and 100ul of 1mg/mL dansyl chloride in acetone. Vortexed samples were then heated to 60°C to complete the derivatization. RESULTS Initial injections made with the generic gradient showed similar separations on the BEH 2-EP and BEH Amide columns. The BEH Amide column had somewhat better separation between 17epiestriol and 16Epiestriol. The overall run time was shorter on the BEH 2-EP and this column was selected for optimization. The separation was optimized with the following co-solvent gradient method: Time % Modifier B (methanol) Initial 4 0.5 4 1.75 12 2.9 12 3.2 25 3.4 25 3.5 4 4.0 4 (column re-equilibrated) Figure 4 shows separations on both columns using the same optimized gradient profile. The high linear velocity of the mobile phase and small column volume allows rapid column re-equilibration and a four minute run time on the BEH 2-EP. ACQUITY UPC2 BEH 2-Ethylpyridine 2.1x50mm 1.7um Figure 4 Separations by BEH Amide and BEH 2-EP columns. ACQUITY UPC2 BEH Amide 2.1x50 1.7um Source Temp 150°C Desolvation Temp 300C Desolvation Flow 800 L/hr Cone Gas Flow 150 L/hr Capillary Voltage 3kV Makeup flow 0.1% formic acid in methanol 200 uL/min Experiments were made to determine optimal mass spec source conditions including capillary voltage, makeup flow and composition. They are: Sensitivity A series of dilutions of the 50 pg/uL mixture was made and derivatized to determine linearity and limits of quantitation for neat standards. Figure 5 shows chromatograms for the limit of quantitation of E1, E2 and E3. The limit of quantitation based on the signal to noise for E1, E2 and E3 is 0.02 ng/mL or 0.02 pg injected on column. The estimated limit of quantitation of 16 and 17 Epiestriol is 0.04 ng/mL. Figure 6 Chromatograms at the limit of quantitation for E1, E2 and E3. Linearity Good linearity is demonstrated from 50 ng/mL to 0.02 ng/mL for the metabolites as shown in Figures 7 – 11. Figure 10 16 Epiestriol calibration from 0.02 ng/mL to 50 ng/mL. Figure 11 17 Epiestriol calibration from 0.02 ng/mL to 50 ng/mL. Figure 7 E1 calibration from 0.02 ng/mL to 50 ng/mL. Figure 8 E2 calibration from 0.02 ng/mL to 50 ng/mL. Figure 9 E3 calibration from 0.02 ng/mL to 50 ng/mL. Figure 2 Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland to download a copy of this poster, visit www.waters.com/posters
