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METHOD DEVELOPMENT FOR THE SEPARATION OF SULFATED ESTROGENS BY ULTRA-PERFORMANCE

CONVERGENCE CHROMATOGRAPHY (UPC2)

Christopher J. Hudalla1, Kenneth J. Fountain1, Manisha A. Patel2,Mark A. Hardink2 and Frank W. Riley2

(1) Waters Corporation, Milford, MA USA, (2) Pfizer Inc., Groton CT USA

INTRODUCTION

The chromatographic analysis of steroids and

steroid derivatives often presents a difficult

challenge due to their structural similarities. One

such group of compounds is the conjugated

estrogens. The sulfated estrogens are frequently

used as hormone replacement therapy for post-

menopausal women and for younger women with

hormone deficiencies.1,2 Because of these

treatment regimes, there is great interest in the

development and characterization of therapeutic

preparations of the sulfated estrogens (structures

shown in Figure 1).

Estrone (1)MW = 350.43

OCH3

O

SO O

OH

HH

H

Equilin (2)MW = 348.41

OH

OCH3

O

SO O

HH

-8,9-Dehydroestrone (3)MW = 348.41

OCH3

O

SO O

OH

H

Equilenin (4)MW = 346.41

OCH3

O

SO O

OH

17-Estradiol (5)MW = 352.45

OHCH3

O

SO O

OH

HH

H

17-Estradiol (6)MW = 352.45

OHCH3

O

SO O

OH

17-Dihydroequilin (7)MW = 350.43

OHCH3

O

SO O

OH

HH

17-Dihydroequilin (8)MW = 350.43

OHCH3

O

SO O

OH

HH

17-Dihydroequilenin (9)MW = 348.41

OH

OHCH3

O

SO O

H

17-Dihydroequilenin (10)MW = 348.41

OHCH3

O

SO O

OH

H

Figure 1. Structures of ten sulfated estrogens. Compounds with the same

color font for molecular weight indicate that they are isobaric. The

numbers assigned to each compound are used for peak identification in all

figures.

Additional optimization

Explore gradient slope

Adjust modifier concentrations and gradient times (tg)

Employ flow gradient to maximize peak capacity

Explore effects of pressure, temperature and flow rate

Additive Screening:ammonium hydroxide, isopropyl amine, ammonium acetate,

ammonium formate, formic acid, water

Initial Screening of Stationary Phases and Modifiers:BEH, BEH 2-EP, HSS C18 SB

methanol, ethanol, and acetonitrile

Figure 3. Method development process for the UPC2 separation of the

sulfated estrogen mixture.

METHODS

Sample:

A mixture of the synthetic sulfated estrogens was prepared

from stock solutions with final concentrations between 0.05-0.4 mg/mL, using ethanol as a diluent.

INITIAL METHOD DEVELOPMENT

Initial method development was performed using a 5-minute generic screening gradient from 5-50% modifier on three

different stationary phases to determine which would provide the best separation. Modifier screening was also performed

using methanol, ethanol and acetonitrile. Because of the charged nature of these analytes, additional additives were

required for analyte elution from the column. For the initial screening conditions, 10 mM ammonium acetate and 5% water

was included in each modifier. Results from the column/modifier screening are shown in Figure 4.

COLUMNS SCREENED:

ACQUITY UPC2 BEH, 1.7 µm, 3.0 x 100 mm

ACQUITY UPC2 BEH 2-Ethylpyridine, 1.7 µm, 3.0 x 100 mm

ACQUITY UPC2 HSS C18 SB, 1.8 µm, 3.0 x 100 mm

INITIAL SCREENING CONDITIONS:

Instrument: ACQUITY UPC2 with PDA detection

Mobile Phase A: CO2 (tank, medical grade) Modifier B: methanol, ethanol or acetonitrile

Additive: 10 mM ammonium acetate and 5% H2O Gradient: 5% to 50% Modifier B in 5 minutes

Flow rate: 1.3 mL/min ABPR Pressure: 2175 psi

Column Temp.: 50 °C UV Detection: 220 nm (compensated for 380-480 nm)

Injection Vol.: 1.5 µL

Additional Parameters Evaluated:

Additives, pressure, temperature, flow rate, gradient slope.

The current USP method employing gas chromatography (GC)

requires approximately 3 hours of sample preparation and derivatization, and results in inadequate resolution for one pair

of compounds (Figure 2).3

Additional methods have been developed utilizing LC/MS

yielding a relatively complex method requiring approximately 100 minutes for analysis.4 Here we demonstrate the

application of Convergence Chromatography to this separation, performing method development following a systematic

stepwise approach (Figure 3).

RESULTS

Column/Modifier Screening:

Results from the initial screening experiments are shown in

Figure 4. Based on these results, the ACQUITY UPC2 BEH 2-Ethylpyridine, 1.7 µm, 3.0 x 100 mm Column was chosen for

additional method optimization using methanol as the modifier.

Minutes2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

HSS C18 SBa) ACN

b) EtOH

c) MeOH

Minutes2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

BEHa) ACN

b) EtOH

c) MeOH

Minutes2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

BEH 2-EPa) ACN

b) EtOH

c) MeOH

Figure 4. Screening of the sulfated estrogen mixture on ACQUITY UPC2,

3.0 x 100 mm Columns: 1.8 µm HSS C18 SB (top), 1.7 µm BEH (middle)

and 1.7 µm BEH 2-Ethylpyridine (bottom), using ACN (a), EtOH (b), and

MeOH (c) mobile phase modifiers, each with 10 mM ammonium acetate and

5% water as additive.

0.1% FA25mM AmFor

in MeOH

10mM AmOAc5% H2Oin MeOH

0.3% NH4OH5% H2Oin MeOH

0.3% NH4OHin MeOH

0.3% IPAminein MeOH

a)

b)

c)

d)

e)

0.20

0.40

0.60

0.00

0.20

0.40

0.00

0.20

0.40

0.00

0.20

0.40

AU

AU

AU

AU

AU

0.00

0.20

0.40

Minutes3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00

1 2

3

4 56

78

9 10

Figure 5. Screening of the estrogen mixture using a methanol modifier

with various additives: 0.1% formic acid and 25 mM ammonium formate

(a), 10 mM ammonium acetate and 5% water (b), 0.3% ammonium

hydroxide and 5% water (c), 0.3% ammonium hydroxide (d), and 0.3%

isopropyl amine (e).

AU

0.00

0.20

0.40

AU

0.00

0.20

0.40

AU

0.00

0.20

0.40

AU

0.00

0.20

0.40

AU

0.00

0.20

0.40

Minutes

2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

Rs(6,7) = 0.89

ABPR

1700 psia)

b)

c)

d)

e)

Rs(6,7) = 0.96

Rs(6,7) = 1.00

2175 psi

2500 psi

2900 psi

3200 psi

12

3

4 5 6

78

9 10

Figure 6. Screening of the estrogen mixture with variable ABPR settings:

1700 psi (a), 2175 psi (b), 2500 psi (c), 2900 psi (d), and 3200 psi (e).

AU

0.00

0.10

0.20

0.30

AU

0.00

0.10

0.20

0.30

AU

0.00

0.10

0.20

0.30

Minutes2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40

30°C

40°C

50°C

Rs(6,7) = 1.09

Rs(6,7) = 1.03

Rs(6,7) = 1.00

1

2

3

4 5 6

7

8

910

Figure 7. Evaluation of the estrogen mixture at various temperatures: 30°

C (top), 40°C (middle), and 50°C (bottom). A 5-minute gradient from

9-40% modifier was run at 1.3 mL/min with an ABPR setting of 3200 psi.

AU

0.00

0.20

AU

0.00

0.20

AU

0.00

0.20

AU

0.00

0.20

AU

0.00

0.20

AU

0.00

0.20

AU

0.00

0.20

Minutes1.60 2.00 2.40 2.80 3.20 3.60 4.00 4.40 4.80 5.20 5.60 6.00 6.40 6.80

RS(6,7) = 0.94

RS(6,7) = 0.97

RS(6,7) = 1.01

RS(2,3/6,7) = 0.94/1.06

RS(2,3/6,7) = 0.93/1.04

RS(2,3/6,7) = 0.99/1.10

RS(2,3/6,7) = 1.04/1.13

APBR = 2000 psi

Flow Rate = 1.9 mL/min

tg = 3.4 min

APBR = 2300 psi

Flow Rate = 1.7 mL/min

tg = 3.8 min

APBR = 2600 psi

Flow Rate = 1.6 mL/min

tg = 4.1 min

APBR = 3200 psi

Flow Rate = 1.3 mL/min

tg = 5.0 min

APBR = 3500 psi

Flow Rate = 1.2 mL/min

tg = 5.4 min

APBR = 3800 psi

Flow Rate = 1.0 mL/min

tg = 6.5 min

APBR = 4100 psi

Flow Rate = 0.8 mL/min

tg = 8.1 min

a)

b)

c)

d)

e)

f)

g)

Figure 8. Screening of the estrogen mixture with variable flow rates and

ABPR settings. The settings for ABPR, flow rate, and tg are: 2000 psi, 1.9

mL/min, 3.4 min (a), 2300 psi, 1.7 mL/min, 3.8 min (b), 2600 psi, 1.6 mL/

min, 4.1 min (c), 3200 psi, 1.3 mL/min, 5.0 min (d), 3500 psi, 1.2 mL/min,

5.4 min (e), 3800 psi, 1.0 mL/min, 6.5 min (f), and 4100 psi, 0.8 mL/min,

8.1 min (g). The gradient was 9-40% modifier at 30°C.

Additive Screening:

Additives have been shown to play a critical role in the

retention mechanisms governing separations with liquid CO2, with several hypotheses as to their function: suppression of

ionization, formation of ion pairs, coverage of stationary phase active sites, or altering the polarity of the stationary and/or

mobile phases. For the current method development, several different additives were explored in combination with a

methanol modifier as shown in Figure 5.

Effects of Temperature and Pressure:

Based on the evaluations shown above, 0.3% isopropyl amine

was chosen as additive to explore the effects of temperature and pressure on the separation (Figures 6 and 7). It is clear

from the chromatography above that there are two critical peak pairs: 2 & 3 and 6 & 7. The resolution of these peaks,

when possible, was used to asses any improvements in the separation.

Effects of Flow Rate:

Because of the relationship between flow rate and pressure, it

is impossible to study flow rates without including a pressure

component. For these evaluations, at each pressure setting,

the flow rate was set to the maximum flow possible while

staying within the pressure specifications of the UPC2 system.

For each set of conditions, the gradient time (tg) was adjusted

to maintain the same gradient volume (13.9 column volumes)

for each separation.

References

1 National Institutes of Health. State-of-the-Science Conference

statement: management of menopause-related symptoms. Ann

Intern Med., Jun 21 2005;142(12 Pt 1):1003-13.

2 Ettinger B, Ensrud KE, Wallace R, et al. Effects of ultralow-dose

transdermal estradiol on bone mineral density: a randomized clinical

trial. Obstet Gynecol., Sep 2004;104(3):443-51.

3 USP Monograph. Conjugated Estrogens, USP35-NF30 [5481]. The

United States Pharmacopeial Convention, official from August 1,

2012.

4 Xu, X., et al. Measuring Fifteen Endogenous Estrogens Simultaneously

in Human Urine by HPLC/MS., Anal. Chem., 2005; 77:6646-6654.

CONCLUSION

This application highlights the ability of UPC2 to

address challenging separations of structurally

similar compounds making it well suited for the analysis of steroids and steroid-related compounds.

The ability to analyze sulfated estrogens in their

native form without the need for desulfation and

derivatization greatly reduces sample preparation time.

Method development was carried out using a

systematic stepwise approach resulting in a final

method meeting all requirements with resolution values (Rs) > 1.5 for each of the ten sulfated

estrogens.

Exploration of critical method parameters included

choice of column chemistry, modifier, additive, pressure, temperature, flow rate and gradient slope.

Application of UPC2 technology to the separation of

the synthetic sulfated estrogen mixture results in

>.90% reduction in analysis time relative to the current USP method using gas chromatography.

FINAL OPTIMIZED CONDITIONS:

The separation still did not meet the specified resolution

requirements of Rs > 1.5 for each analyte. In previous evaluations, improvement in resolution had been observed

with lower temperatures and shallower gradients. As part of additional optimization, both of these effects were explored

further, with the resulting conditions and chromatography shown below in Figure 12.

Instrument: ACQUITY UPC2 with PDA detection

Column: ACQUITY BEH 2-Ethylpyridine

Configuration: 3.0 x 100 mm, 1.7 µm

Mobile Phase A: CO2 (tank, medical grade)

Modifier B: Methanol (Fisher Optima® grade)

Additive: 0.3% ammonium hydroxide

Gradient: 8% to 27% Modifier B in 12 minutes

Flow Gradient: from 1.4 to 0.9 mL/min

ABPR Pressure: 4100 psi

Column Temp.: 20 °C

UV Detection: 220 nm (compensated for 380-480 nm)

Injection Vol.: 1.5 µL

Minutes

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00

AU

0.00

0.04

0.16

RS(2,3/6,7)

1.65/1.52

1

2

3

45

6

7

8

9

10

0.08

0.12

Figure 12. Separation of the sulfated estrogen mixture using the final

optimized parameters. Resolution requirements with Rs > 1.5 were met for

all analytes, with values of 1.65 and 1.52 for the two critical peak pairs,

peaks 2-3 and peaks 6-7, respectively.

Gradient Slope:

The slope of the gradient can also have significant impact on

the separation. The gradient slope can be explored by altering

either the beginning and ending modifier concentrations or by

increasing or decreasing the gradient time (tg). This is

demonstrated in Figures 10 and 11.

Flow Gradients:

Flow gradients were used to maximize the efficiency of the

separation, within the pressure specifications of the UPC2

instrument. As the gradient transitions from 9-40% modifier,

the flow rate decreases linearly from 1.3 to 0.8 mL/min. The

gradient time (tg) was adjusted to maintain the same gradient

volume as in the constant flow example (13.9 column

volumes). Figure 9 shows the comparison of the separation

with and without the flow gradient.

Figure 10. Evaluation of gradient slopes adjusted by changing the

beginning and ending modifier concentrations: 8-33% (top), 6-35%

(middle), and 4-37% (bottom). For each experiment, the gradient time

(tg) was 6.2 minutes (13.9 column volumes). AU

0.00

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Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

RS(2,3/6,7)

1.21/1.20

8-33%1.3-0.8mL/min

6-35%1.3-0.8mL/min

4-37%1.3-0.8mL/min

RS(2,3/6,7)

1.19/1.17

RS(2,3/6,7)

1.15/1.14

12

3

45

6

7

8

910

Figure 11. Evaluation of an 8-33% modifier gradient with slopes adjusted

by changing the gradient time (tg): 5 minutes (a), 7 minutes (b), 9 minutes

(c), and 12 minutes (d). The gradient volumes were 12.3, 17.3, 22.2, and

29.6 column volumes, respectively.

RS(2,3/6,7)

1.14/1.16tg = 5 min

(12.3 cv)

AU

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0.20

AU

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0.20

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0.00

0.20

Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

RS(2,3/6,7)

1.25/1.23

RS(2,3/6,7)

1.33/1.25

RS(2,3/6,7)

1.43/1.27

tg = 7 min

(17.3 cv)

tg = 9 min

(22.2 cv)

tg = 12 min

(29.6 cv)

a)

b)

c)

d)

12

34 5 6

7 89 10

Figure 9. Comparison of gradient separations using 9-40% modifier at a

constant flow rate of 0.8 mL/min (top) with the separation employing a

flow gradient from 1.3 to 0.8 mL/min (bottom). The system pressure

trace for each separation is shown in orange.

Time(min)

Flow(mL/min)

% CO2 %B Curve

0.0 0.8 91 9 -

8.1 0.8 60 40 6

8.9 0.8 91 9 11

13.0 0.8 91 9 11

Time(min)

Flow(mL/min)

% CO2 %B Curve

0.0 1.3 91 9 -

6.2 0.8 60 40 6

8.0 0.8 91 9 1

8.9 1.3 91 9 6

13.0 1.3 91 9 6

psi

4800

5000

5200

5400

5600

psi

4800

5000

5200

5400

5600

9-40%0.8mL/min

9-40%1.3-0.8mL/min RS(2,3/6,7) = 1.12/1.20

RS(2,3/6,7) = 1.04/1.13

AU

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AU

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Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Figure 2. GC-FID separation of ten estrogens using the USP method for

conjugated estrogens. Two of the compounds, peaks 6 and 7, are not

completely resolved (circled in red).

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30 35

Mv

Retention Time (min)

5

6

7

89 10

1

2

3 4

InternalStandard

17-Estradiol (6)MW = 352.45 OHCH3

O

SO O

OH

17-Dihydroequilin (7)MW = 350.43 OHCH3

O

SO O

OH

HH