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INTRODUCTIONEstrogen 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 DePhillipo1Waters 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 UPC2 MS/MS for Clinical Research Christopher J. Hudalla1, Stuart Chadwick2, Fiona Liddicoat2, Andrew Peck1 and Kenneth J. Fountain1 1 Waters Corporation, Milford, MA USA, 2 Waters Corporation, Manchester, UK

CONCLUSIONS

UltraPerformance Convergence Chromatography provides arapid four minute separation of five key estrogenmetabolites, including three isobaric species. The efficiencyof UPC2 creates a separation which is faster thanconventional supercritical liquid chromatography or highperformance liquid chromatography.

The sensitivity of this method using UPC2 and tandemmass spectrometry is in the range of sub-picogramsinjected on column for neat estrogen standards derivatizedwith dansyl chloride.

The ability of this method to quantitate an expanded panelof metabolites in urine must be explored to determine it'spotential 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 UPC2, to separate estrogen metabolites.

UPC2 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 UPC2 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 UPC2 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 protocols1. 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 UPC2 BEH 2-Ethylpyridine 2.1x50mm 1.7umACQUITY UPC2 BEH Amide 2.1x50 1.7um

Screening Conditions:

Instrument: ACQUITY UPC2

Xevo TQ-S MS515 makeup pump

Mobile Phase A: Liquid CO2 (beverage grade)

Modifier B: MethanolNeedle Wash: 70/30 MeOH/IPAFlow Rate: 2.5 mL/minGradient: 5% to 25% Modifier B in 3 minutes.

equilibration at 5% modifier B for 1 min.

Column Temp: 60°CCCM Pressure: 1800 psiData System MassLynx 4.1MakeUp 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.

RESULTSInitial 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 40.5 41.75 122.9 123.2 253.4 253.5 44.0 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

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