Maximizing Sensitivity in the LC-MS/MS Determination of ... · PDF fileEthynyl Estradiol (EE)...

Jonathan Bailey, Emmanuelle Caron, Allan J. Cessna, Annemieke Farenhorst and Claudia Sheedy
Western Canada Trace Organics WorkshopCalgary, Alberta
April 23-25, 2007
Maximizing Sensitivity in the LC-MS/MS Determination of
Estrone (E1), 17-Estradiol (E2), Estriol (E3) and 17-Ethynyl Estradiol
in Natural Waters

Male Fish Feminization linked to WW

Saskatoon Wastewater
Treatment Plant

EstrogensOH
OH
HO
HH
H
Estriol E3
HO
O
H
H H
Estrone E1
HO
H
OH
H H
17-Estradiol E2
HO
OH
H H
H
17-Ethynyl estradiol EE

Method Development Plan
1. Solid-phase extraction using Oasis HLB cartridges (~100x concentration)
2. Reverse-phase liquid chromatographic separation using C18 column
3. Mass Analysis using ESI(-) LC/MS/MS

4 Estrogens using ESI(-) [10 g/L]E1_E2_E3_EE_10ppb_50Meoh_50h20_10uLNH4OH_100uL
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
%
0
100April14_07_Sample20 4: MRM of 2 Channels ES-
295 > 1451.26e4
Area
2.221416
1.0433
0.4035
0.6115
0.842
1.1427
1.2830 1.85
221.4810
1.618
2.3870
4.03423.1222
2.858
2.654
3.4914
3.266
3.737
3.891
4.407
4.3012 4.675
April14_07_Sample20 3: MRM of 2 Channels ES- 287 > 171
1.82e4Area
1.912729
0.4667 1.0763
1.3731
2.5537
2.03e4Area
2.242205
1.7361
1.3320
1.1624
0.565
0.8612
0.734
1.0613 1.466
1.9034
2.6165
4.0544
3.2414
3.5813
3.415
3.888
4.5546
4.286
4.383 4.7510
1.02e5Area
2.2611665
0.82177
0.5894
0.4832 1.02182
1.7979
1.2675
2.83159
3.94139
E1
E2
E3
EE
11665
2205
2729
1416

(m/z 271) Precursor Ion
95%
-O
OH
H
H H
H
(m/z 145) Fragment Ion
5%
-estradiol fragmentation under ESI(-)
Collision-InducedDissociation
O
H
H(-)

10 ppb ESI(-) Parent IonE1_E2_E3_EE_10ppb_50Meoh_50h20_10uLNH4OH_100uL
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
295 > 2953.41e5
Area
2.1838123
1.983127
1.813347
1.011833
0.17684
1.111334
1.28991 2.551452
2.75323
3.76363
3.06108
4.57523
3.93138
4.80141
2.90e5Area
1.9147525
2.28664
4.36e5Area
2.2448187
1.935743
1.8339831.30
4060.66182
4.12419
2.71412
2.97320
3.48305
3.68131
3.85e5Area
2.2640173
1.9371691.29
7951.162292
0.55532
2.5020494
2.83526
4.71264
E1
E2
E3
EE
40173
48187
47525
38123

ESI(-) TIC (10 ppb) E1_E2_E3_EE_10ppb_50Meoh_50h20_10uLNH4OH_100uL
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
TIC3.53e5
Area
2.1839569
1.983135
1.813363
1.011855
0.17931
0.40323
0.57163
1.111381
1.281011 2.55
14482.75318
3.76369
3.06107
4.57505
3.9368
4.80146
April14_07_Sample20 3: MRM of 2 Channels ES- TIC
3.08e5Area
1.9147741
4.56e5Area
2.2450461
1.9398791.062223
0.86384
0.66183
0.32251
0.22244 0.4668
1.30888
4.12446
2.71419
2.97322
3.48301
3.2846
3.68148
3.8163
4.28202
4.5595
4.87e5Area
2.2652851
1.9373471.29
7401.16237
0.55573
0.82142
2.5019338
2.83642
3.37681
4.71258
E1
E2
E3
EE
52851
50461
47741
39569

100mL_WWTP_afterChlorination_100uL_rep1_istd10uL_10uLNH4OH
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
TIC4.45e6
Area
2.05649140
1.04e52.111.81
1.711.61
1.100.06 0.20
0.53
1.30
1.87
2.412.61
2.78 3.493.383.12 3.75 3.95 4.12 4.494.32 4.894.69
1.24e51.83
1.30
1.200.99
0.560.22 0.46
1.401.60
1.93
2.242.34
2.50 2.64 2.74 2.91 3.613.08 3.513.34 3.913.85 4.28 4.724.45
1.45e51.96
1.050.990.380.15 0.62
1.631.521.29
2.732.30 2.43
4.854.013.273.143.003.94
3.51 3.74 4.24 4.584.34 4.71
Ethynyl Estradiol (EE) present in effluent
E1
E2
E3
EE 649140

Ethynyl Estradiol (EE) present in effluent 100mL_WWTP_afterChlorination_100uL_rep1_istd10uL_10uLNH4OH
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00
%
0
295 > 1452.73e4
Area
2.053547
0.9743
0.1097
0.7428
1.6537
3.4266
2.38121
4.3693
4.5034
4.42e6Area
2.05645667
4.45e6Area
2.05649140
649140
645667
3547
Combined TIC
m/z 295>295
m/z 295>145

Summary of ESI(-) LC/MS/MS
10.0 g/L instrumental detection limit Sum of daughter ion and parent ion
traces increases sensitivity but costs specificity
100L injection volume increases sensitivity but reduces column life
Method detection limit = 100 ng/L based on 100x conc. from SPE)

Where do we go from here? ESI(-) LC/MS/MS provided evidence of
ethynyl estradiol (EE) in Saskatoon wastewater effluent at ~ 4000 ng/L
Biological activity threshold for estrogens in the enviroment is ~ 1 ng/L
Need lower method detection limit to assess environmental impact of EE

The Box

Challenge: connect all 9 dots using continuous straight-lines

Puzzle?

Eureka! Early literature suggested using dansyl
chloride derivatization to boost selectivity and sensitivity in LC/UV methods for estrogens
Recent literature indicated a 1000x increase in sensitivity for dansyl-derivatives using tandem mass spectrometry in ESI(+) over ESI(-)

Derivatization of ethynyl estradiol
S
O
OCl
N
HO
OH
H H
H
S
O
O
N
O
OH
H H
H
+
Dansyl chloride 17-Ethynyl estradiol
dansyl-ethynyl estradiol
60oC (100mM carbonate pH 11)

Derivatization Procedure
Add 150uL dansyl chloride (in acetone) and 50uL sodium carbonate buffer (pH 11) to the dry residue obtained after either li

Maximizing Sensitivity in the LC-MS/MS Determination of ... · PDF fileEthynyl Estradiol (EE)...

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