A Rapid LCMS Method for Evaluation of EPA 1694 and 6810 Contaminants in Drinking Water

Rachel A Lieberman , Jeremy Post , Jonathan Edwardsen , William Lipps , Christopher Gilles , Shimadzu Scientic Instruments, Columbia, MD, USA
1

1 1

NOVEL ASPECT

RESULTS - CHROMATOGRAMS

An ultra-fast polarity switching ve minute method to analyze

and quantify many different contaminants of emerging concern
(CECs) by LCMS-MS.

INTRODUCTION
Contaminants of emerging concern (CECs) are chemicals such as
endocrine disrupting compounds, pharmaceuticals and personal
care products that have been detected in the environment.
Most of these chemicals enter the environment through treated
and raw sewage and are detrimental to the aquatic organisms at
picogram per milliliter levels. Early detection for these CECs was
performed using gas chromatography mass spectrometry
(GC-MS) but more recently liquid chromatography mass spectrometry (LC-MS) has become more popular for detecting low
levels of these contaminants in wastewater. This poster describes a comprehensive method for evaluating multiple steroids, pharmaceuticals and personal care products at picogram
per milliliter levels in drinking water using ultra-fast LCMS-MS.

Area

Analyte
Acetaminophen
Trimethoprim
Ciprooxacin
Primidone
Sulfamethoxazole
Caeine
Fluoxetine
Erythromycin
Carbamazepine
Cortisol
Cortisone
17a-Ethynylestradiol

Ret. Time
0.562
0.991
1.443
1.445
1.598
1.609
2.541
2.581
2.869
2.87
3.053
3.146

Analyte
Ret. Time
Aldosterone
3.32
Diclofenac
3.538
11-Deoxycortisol
3.552
DHEA
3.635
Estrone
3.635
Gembrozil
3.655
Triclosan
3.768
Testosterone
3.869
Androstenedione
4.214
11-Deoxycorticosterone
4.233
17-hydroxyprogesterone 4.233
Progesterone
4.558

10:237.30>194.20(+)
50000 10:237.30>193.05(+)

Carbamazepine

20000000

40000

5500000
15000000

30000

5000000

20000

10000000

4500000

10000

5000000
0

4000000
0
0.0

3500000

2.750

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

Sulfadimethoxazole

Level
11
12
13
14
15
16
17
18
19
20

Calibration levels

ng/mL (ppb)
1.25
0.7813
0.6250
0.2930
0.1953
0.1465
0.0977
0.0781
0.0488
0.0391

2.875

2.900

2.925

2.950

2.975

2000
1500

2000000

1500000

1000

1000000

1000000

500000

500
0
1.500

10

20

30

40

50

60

70

80

90

1.525

1.550

1.575

1.600

1.625

1.650

1.675

1.700

Conc.

Area

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

min

6000 1:152.25>110.15(+)
1:152.25>64.95(+)

Acetaminophen

7500000

5000

4000

5000000
3000

900000

2000

2500000
1000

800000

A representative chromatogram for a 10 ppb

standard of the analyte mix is shown. Carbamazepine has the highest response as shown
in the top gure. All other analytes are
shown in the zoomed in region from 0 100000 intensity in the bottom gure. The
retention times for each of the analytes are
listed in the table above.

700000

10

20

30

40

50

60

70

80

90

600000

1000

2000000

Cortisone

500000

- Ultrafast MRM methods

- Up to 555 MRM transitions per sec
- Heated electrospray source
- Scan speeds up to 30,000 u/sec
- Polarity switching 5 msec

LCMS-8060 Parameters
Nebulizing Gas Flow
3 L/min
Heating Gas Flow
13 L/min
Drying Gas Flow
7 L/min
Interface Temperature
350 C
DL Temperature
125 C
Heat Block Temperature
350 C

291.15>230.20
332.15>288.15
219.25>91.05

Sulfamethoxazole

254.20>92.05

Caeine
Fluoxetine
Erythromycin
Carbamazepine
Cortisol

195.30>138.10
310.30>44.10
734.50>576.35
237.30>194.20
363.30>121.10

Cortisone

361.20>163.10

17a-Ethynylestradiol

295.20>144.95
361.20>343.25
294.00>250.10
347.35>109.10
271.25>133.05
269.20>145.10
249.30>121.00
287.10>34.95

Testosterone

289.25>97.05

Androstenedione

287.35>97.10

11-Deoxycorticosterone 331.20>97.10
17-hydroxyprogesterone 331.20>97.00
Progesterone

0.650

0.675

12:361.20>163.10(+)
12:361.20>121.20(+)

750

2.950

200000

100000

Area

Trimethoprim

Gembrozil
Triclosan

0.625

500000

10

20

30

40

50

60

70

80

90

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

min

315.40>109.10

Reference Ions Ret. Time

152.25>64.95;
0.562
152.25>93.05
291.15>261.05;
0.991
291.15>123.20
332.15>231.10;
1.443
332.15>314.20
219.25>162.30;
1.445
219.25>119.05
254.20>108.10;
1.598
254.20>156.20
195.20>110.05
1.609
310.30>148.30
2.541
734.50>116.15
2.581
237.30>193.05
2.869
363.30>327.10
2.87
361.20>121.20;
3.053
361.20>105.15
295.20>159.20;
3.146
295.20>267.15
361.20>315.25;
3.32
361.20>325.00
3.538
347.35>97.00;
3.552
347.35>253.25
271.25>159.20;
3.635
271.25>253.15
269.20>143.10;
3.635
269.20>159.15
3.655
3.768
289.25>109.15;
3.869
289.25>79.15
287.35>109.15;
4.214
287.35>79.20
331.20>109.10
4.233
331.20>109.10
4.233
315.40>97.05;
4.558
315.40>79.15

3.000

3.025

3.050

3.075

3.100

3.125

3.150

700 22:295.20>144.95(-)
22:295.20>159.20(-)
600
500

40000

0.75

2.975

Conc.

17a-Ethynylestradiol

50000

152.25>110.15

Estrone

0.600

Acetaminophen

DHEA

0.575

1000000

300000

m/z

11-Deoxycortisol

0.550

250

Analyte

Diclofenac

0.525

500

400000

400

30000

300
200

20000

100
0
3.050

10

20

30

40

50

60

70

80

90

3.075

3.100

3.125

3.150

3.175

3.200

3.225

3.250

Conc.

Limit of Quantitation* - All Analytes in Neat Standard

RESULTS - DRINKING WATER

Aldosterone

0.500

1500000

LCMS-8060 Parameters

Primidone

0.475

Area

Ciprooxacin

0.450

Conc.

10000

ng/mL (ppb)
100
75
50
37.5
25
18.75
12.5
10
5
2.5

2.850

2500

2000000

LC Chromatogram at 10 ppb for all twenty four compounds in under 5 minutes.

Level
1
2
3
4
5
6
7
8
9
10

2.825

3000 5:254.20>92.05(+)
5:254.20>156.20(+)

3000000

Zoom

2.800

Conc.

4000000

2500000

Retention times for all analytes analyzed in the mixture.

2.775

Area

3000000

0.50

A mixture of steroids (estrone, progesterone, testosterone, 17

-ethynylestradiol, aldosterone, 11-deoxycortisol, 11-deoxycorticosterone, cortisol, cortisone, 17-hydroxyprogesterone, androstenedione, DHEA), pharmaceuticals and personal care products (ibuprofen,
triclosan, sulfamethoxazole, caffeine, acetaminophen, erythromycin,
uoxetine, gembbrozil, diclofenac, ciprooxacin, carbamazepine,
primidone, trimethoprim) have been analyzed using ultra-fast liquid
chromatography mass spectrometry. Heated electrospray ionization
(hESI) in both positive and negative modes with polarity switching
was used for ionization of the compounds. MRM transitions for each
analyte were optimized and separated using reversed phase chromatography in a single ve minute method. Tap water was directly injected in triplicate for the drinking water samples.
Mobile Phase A: Water with additive
Mobile Phase B: 0.3% formic acid in Methanol
Column: Restek Raptor Biphenyl (2.7 m x 100 x 2.1mm)
Gradient: Linear from 34% - 100% B.
Flow Rate: 0.6 mL/min
Column oven: 40C
Injection volume: 1 L

Calibration Curve and MS Chromatograms

Chromatogram for EPA Analysis

METHODS

Chromatography Conditions

RESULTS - CALIBRATION CURVES

Analyte
Acetaminophen
Trimethoprim
Ciprooxacin
Primidone
Sulfamethoxazole
Caeine
Fluoxetine
Erythromycin
Carbamazepine
Cortisol
Cortisone
17a-Ethynylestradiol

12500

10000

7500

5000

2500

Ret. Time
0.562
0.991
1.443
1.445
1.598
1.609
2.541
2.581
2.869
2.87
3.053
3.146

ng/mL (ppb)
100 - 0.0977
100 - 0.625
100 -10
100 - 0.625
100 - 0.0977
100 - 0.0977
37.5 - 2.5
100 - 5
25 - 0.0391
100 - 0.0977
100 - 0.0977
100 - 0.781

Analyte
Ret. Time
Aldosterone
3.32
Diclofenac
3.538
11-Deoxycortisol
3.552
DHEA
3.635
Estrone
3.635
Gembrozil
3.655
Triclosan
3.768
Testosterone
3.869
Androstenedione
4.214
11-Deoxycorticosterone
4.233
17-hydroxyprogesterone 4.233
Progesterone
4.558

ng/mL (ppb)
100 - 0.0977
100 - 0.0977
100 - 0.0977
100 - 2.5
100 - 0.292
100 - 0.292
100 - 0.781
100 - 0.0488
100 - 0.0977
100 - 0.0977
100 - 0.0977
100 - 0.0977

*LOQ for the analytes measured in neat standard. More work needs to be completed to give comprehensive results in matrix.

0
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

min

LC Chromatogram for drinking water sample. Most analytes are not detected.

291.15>230.20(+)
291.15>261.05(+)
900 291.15>123.20(+)

734.50>576.35(+)
734.50>116.15(+)
2000

800

310.30>44.10(+)
310.30>148.30(+)
1500

Analysis of the analytes in drinking water

sample. Direct injection of a 1 L sample with
no sample preparation.

1750

700

1250

1500

600

1000

1250

500

1000

400

750

750

Most analytes are not detected, however,

erythromycin, trimethoprim and uoxetine are
detected.

500

300

500

200

250

250

100

0
0.9

1.0

1.1

Trimethoprim
1.6 ppb

0
2.5

2.6

Erythromycin
3.4 ppb

2.5

2.6

Fluoxetine
3.2 ppb

CONCLUSIONS
A robust and rapid ve minute method for evaluating multiple
endocrine disrupting compounds, pharmaceuticals and personal
care products was developed using ultra-fast liquid chromatography mass spectrometry. Most analytes were detected in the parts
per trillion (pg/mL) range in neat standard. Low levels of a few
compounds were detected in the drinking water sample. Furthermore, this method can be applied to other environmental water
samples.
For research purposes only. Not for use in diagnostic procedures.