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Environmental
Figure 3 An Allure" Biphenyl column provides supe rior selectivity
and retention for steroids (UV detection).
1
Columns: 150mm x4.6mm,3f.1m
Mobile phase: water:acetonitrile, 50:50
Temperature: ambient
2 3 Flow rate: 1.5ml /min.
Detection: UV, 254nm
Allure8 Biphenyl column
Excellent resolution,
including isomersl
'--J U U \
LC_BP0385
I i I i i I I I
1.0 2.0 2.0 '.0 5.0 '.0 10 '.0 '.0 111.0
M,n.
4,7
1. estriol
2. 17B-estradiol
3. l7a-estradiol C18 Column
4. 17a-ethynyl estradiol
5. testosterone
6. estrone
7. norethindrone
u
I
20
y"
To mon itor steroid sex hormones in water, we first
developed an extraction procedure, using styrene­
divinylbenzene solid phase extraction disks and
methyl tert-butyl ether (MTBE) as the extraction
solvent. We conditioned the extraction disks with
acetonitr ile and MTBE to remove any pot ential
interferences. After rinsing the disk with distilled
water and loading the disks with one liter of sam­
ple we used lOmL of MTBE to elute the sample.
Prior to analysis the final lOmL extract was con­
centrated to 2mL and exchanged to acetonitrile.
We recognized that the complexity of environmen­
tal samp le mixtures and matrices often would
make difficult a complete chromatographic separa­
tion of the steroid sex hormones by HPLC, and
qualitative detection with a non-selective detector
(UV-Vis). Mass spectrometry, with secondary sep­
aration based on m/z, increased our confidence in
the qualitative identifications. We selected LECO
Cor poration's Uniquew LC-TOFMS system for its
high data acquisition rate - 100 spectra/sec. The
ChromaTOF® software Peak Find algorithm can
deconvolute closely eluting peaks, and mass can be
determined accurately, to within 5ppm, to calculate
possible molecular formula. Because the ionization
potent ial differs among the groups of steroid sex
hormones, both negative and positive ESI was
used. The estrogens were amenable to negative ESI,
while the androgens and progestogens showed
much greater sensitivity when we used positive ESI
(Figure 4). We believe this difference is because of
the differing functional groups at position 3.
Figure 4 Sensitive analysis of steroids, using an Allure '" Biphenyl
column, and LECO Unique'"TOFMS.
estrone
450
400
350
300
250
200
150
100
50
600
500
400
300
200
100
estriol
A....
0:50
ethy"yl estradiol
17-a-estradiol
17-Jl-estradiol
Estrogens at Sng on column (-ESI)
Sample:
Inj.: 51ll
Sample diluent: acetonitrile
Column: Allure- Biphenyl
(cal.# 9166352)
Dimensions: 50 x2.1 mm
Particle size: 3f.1~
Pore size: 60A
Conditions:
Mobile phase: A) water + 1:40 2:30 3:20 4:10 5:00 5:50 6:40 1:40
LCEV0404 B) acetonitrile
Time (min.) %B
norethindrone o 45
norgestrel 5 70
17~·methYitestosterone
testosterone
19-nortestosterone
8 70
9 45
15 45
progesterone
1:40 3:20 5:00 6:40 8:20
lCEV0403
Flow: 0.3ml/ min.
Temp.: ambient
Del.: l ecoUnique" TOFMS
Androgens at 1ng on column (+ESI)
These analyses demonstrate that the Allures'
Biphenyl stationary phase, through rt-rt intera c­
tions, offers excellent selectivity for compounds
with unsaturation differences in their hydrocar­
bon ring structures. Additionally, the secondary
separation power of the Unique 's TOFMS system
and Chrom aTOF®software allows overall analysis
time to be reduced, through optimized column
dimensions and run conditions, while qualitative
identification is maintained.
References
1 http:/ /www.epa.gov/ scipoly/ oscpendo/
2 Kuster, M., M.J. lopez, and D. Barcelo, Estrogens and Progesterons in
Wastewater, Sludge, Sediments, andSoil, pp, 3 Handbook of
Environmental Chemistry
http:/ / fantasia/ pdfCache/ 580020.pdf
Allure'" Biphenyl Columns
3J,lm Column, 2.1[Jlm ___-'c"'at-.Lpr!ce_ _
30mm 9166332 $364
50mm 9166352 $364
100mm 9166312 $390
~m Column...i§mm cal # price
30mm 9166335 $364
50mm 9166355 $~
!90mm 9166315 $390
For other column dimensions, and columns with5J1m packing,
please visit our website.
2006.03 800-356-1688 -
• 9 •
Website : E-mail : TelNo : 03 9762 2034 . . . in AUSTRALIA