c) meoh in meoh 25mm amfor sulfated …...convergence chromatography (upc2) christopher j. hudalla 1...
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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
0.10
0.20
0.30
AU
0.00
0.10
0.20
0.30
AU
0.00
0.10
0.20
0.30
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
0.00
0.20
AU
0.00
0.20
AU
0.00
0.20
AU
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
0.00
0.10
0.20
0.30
AU
0.00
0.10
0.20
0.30
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