Optimizing Detection of Steroids inWastewaters Using the Agilent 6490Triple Quadrupole LC/MS System withiFunnel Technology

Author

Neil Cullum

Anglian Water Services

Huntingdon, England

Application Note

Environmental

Abstract

Taking full advantage of the enhanced sensitivity of the Agilent 6490 Triple Quadrupole

LC/MS System requires optimization of method parameters derived on other Triple

Quadrupole platforms. Optimization of steroid analysis methods in the complex matrix

of wastewater results in sensitivity as low as sub-1 ng/L (1 part per trillion).

Introduction

Pharmaceuticals are consumed in high quantities worldwide, and these amountswill continue to increase due to improving health care and longer life expectations.Administered pharmaceuticals are excreted by humans, as a parent compound ormetabolites. They enter sewage treatment plants where they are not entirelyremoved and end up in the environment. These residual pharmaceuticals can negatively impact aquatic and terrestrial ecosystems.

A recent study by the United States Geological Survey (USGS) found chemical cont-aminants in 80% of the streams sampled, and steroids were one of the chemicalgroups most frequently detected. These steroids elicit biological responses at verylow concentrations, including feminization of male fish. A seven year, whole-lakeexperiment in Canada demonstrated that chronic exposure of a fish species to thesteroid 17 alpha-ethynyl estradiol led to a near extinction of that species from thelake [1]. There is also potential for these steroids to travel up the food chain, as wellas into drinking water.

2

The European Union Water Framework Directive(2000/60/EC) [2] promotes sustainable water use, includingthe long-term reduction of wastewater contaminant dis-charges to the aquatic environment, including steroids.Implementation of the Directive requires the development ofsensitive, accurate, and reliable testing methods.

This application note describes the optimization of sensitivemethods for steroid detection on the Agilent 6490 TripleQuadrupole LC/MS System with iFunnel Technology, as partof the Chemical Investigation Program of Directive2000/60/EC. This instrument takes detection limits lowerthan ever, enabling zeptomole level sensitivity at conventionalflow rates, making it an ideal choice for critical pharmaceuti-cal applications, including the detection of steroids in theenvironment. While liquid chromatography/mass spectrome-try (LC/MS) methods for steroid detection and quantificationhave been developed on earlier generation MS instruments,they must be optimized on the 6490 Triple Quadrupole LC/MSSystem in order to maximize sensitivity. Using the optimalparameters determined for detection of several steroid com-pounds, detection limits as low as sub-1 ng/L (one part pertrillion) in wastewaters have been demonstrated.

Experimental

Reagents and StandardsThe ethyl acetate, acetonitrile, propan-2-ol, cyclohexane, andmethanol, were all HPLC grade or glass-distilled. Thehydrochloric acid was 37% analytical reagent grade andammonia solution 30%. The copper nitrate was general pur-pose grade reagent or better. The water was HPLC grade orElga polished water. The styrene divinyl benzene solid phaseextraction (SPE) cartridges (200 mg) were obtained fromBaker. The GF/D glass microfiber filter papers were obtainedfrom Whatman (Kent, UK)

Individual solutions of 100 mg/L of each steroid (estrone,estradiol, and ethynyl estradiol) in acetonitrile were obtainedfrom QMX Laboratories Ltd (Thaxted, UK). A mixed calibrationstandard was prepared by adding each of the individualsteroid solutions to methanol to a concentration of 1.0 mg/L.Working calibration standards were prepared by dilution in90:10 water:methanol to 1, 2, 5, and 10 µg/L. These calibra-tion standards also contained 2 µg/L of each internal standard.

Solid deuterated estrone-D4, estradiol-D5, and ethynyl estra-diol-D4 were obtained from QMX Laboratories for use asinternal standards. Individual 100 mg/L solutions of each

internal standard were prepared in acetronitrile, then a mixedinternal standard was prepared by adding each internal standard to methanol to a final concentration of 1.0 mg/L.A second mix was prepared by diluting an aliquot of the 1.0 mg/mL mix to 0.1 mg/L with methanol.

InstrumentsMethod optimization and analysis of wastewaters were con-ducted on the Agilent 1260 Infinity LC with a 100-µL sampleloop, coupled to the 6490 Triple Quadrupole LC/MS Systemwith iFunnel Technology. Postcolumn addition of 0.1% ammo-nia solution was accomplished using an external pumpattached to a T-junction between the column and the nebu-lizer. The instrument conditions are listed in Table 1.

Sample Collection, Preparation and CleanupSamples were collected in 2-L amber glass bottles and pre-served at the time of sampling with 2 mL of concentratedhydrochloric acid and 0.5 g of copper nitrate and stored atbelow 10 °C. Sample stability under these conditions hasbeen tested up to 14 days after preservation. Once extractionhas taken place, the resulting extracts can be stored for atleast four weeks in a spark-proof refrigerator prior to analysis.

Each sample was filtered through GF/D papers prior to extrac-tion. An aliquot of 20 µL of mixed internal standard wasadded to 1 L of sample. For crude sewage samples, 100 mL ofsample was diluted with 900 mL of water, and 20 µL of mixedinternal standard was added. The sample was then extractedwith the SPE cartridge, which was first conditioned with ethylacetate (5 mL), then methanol (5 mL), then water (3 mL). Thecartridge was loaded with 250 mL of sample, then washedwith 60% methanol (3 mL) followed by water (3 mL). Afterdrying with gas for 40 minutes, the cartridge was eluted with4 mL of ethyl acetate. The extract was evaporated under agentle air stream, with the vial on a 45 °C heating block, andthe residue redissolved in 250 µL of cyclohexane:propan-2-ol(95:5)

The extracts were cleaned up using normal phase chromatog-raphy on an Agilent 1100 series LC with fraction collection onan Agilent ZORBAX Cyano column (p/n 883952-705), 4.6 x 150 mm, 5 µm thermostated at 55 °C, using an isocraticseparation, 95:5 cyclohexane:propan-2-ol at a flow rate of1 mL/min, fraction collection time range 5.8–8.6 minutes. Theextract was evaporated under a gentle air stream, with thevial on a 45 °C heating block, and the residue re-dissolved in250 µL of methanol:water (90:10). With a final volume of250 µL, this is equivalent to a 1000 fold concentration step.Therefore, the calibration standards are equivalent to originalconcentrations of 1, 2, 5, and 10 ng/L.

3

Analysis ParametersThe Agilent Triple Quadrupole LC/MS System analysis parameters are shown in Table 2.

Table 1. LC and MS Instrument Conditions

LC Conditions

Analytical column Agilent C-18 Eclipse Plus, 2.1 × 50 mm, 3.5 µm (p/n 959763-902)

Guard column Luna C-18, 4.0 mm × 2.0 mm

Column temperature 40 °C

Injection volume 25 µL

Mobile phase A = Water

B = Acetonitrile

Run time 16.0 min

Flow rate 0.3 mL/min: 0.1 mL/min postcolumn addition of 0.1% NH3

Gradient program Time (min) % Solvent A % Solvent B

0 90 10

0.5 60 40

10.0 20 80

10.2 0 100

11.5 0 100

11.6 90 10

MS Conditions

Acquisition parameters ESI mode, negative ionization; Dynamic MRM

Sheath gas temperature 300 °C

Sheath gas flow rate 11 L/min

Gas temperature 180 °C

Drying gas Nitrogen, 16 L/min

Nebulizer pressure 45 psig

Nozzle voltage 1,500 V

Vcap voltage 3,000 V

Cell accelerator voltage Varied per optimization study

Delta EMV Varied per optimization study

Low and high pressure ion funnel voltage Varied per optimization study

Scan type dynamic MRM Delta EMV 300 V

Table 2. Time Segments, Transitions, and Fragmentor Voltages used for Optimal Analysis of the Steroid Compounds

Time segmentRetention time(min) Compound

Precursorion

Production CAV

Fragmentorvoltage

Collisionenergy

1 8.0 Estradiol 271.0 145.0 2 380 50

1 8.0 Estradiol-d5 276.0 147.0 2 380 50

1 8.5 EthynylEstradiol 295.0 145.0 2 380 52

1 8.5 EthynylEstradiol-d4 299.0 147.0 2 380 52

1 8.8 Estrone 269.0 145.0 2 380 45

1 8.8 Estrone-d4 273.0 147.0 2 380 45

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Results and Discussion

Optimization of Analysis ParametersWhile methods exist for steroid analysis on previous instru-ments, the analysis parameters for these methods must beoptimized in order to take full advantage of the unique sensi-tivity of the Agilent 6490 Triple Quadrupole LC/MS withiFunnel Technology (6490). In this case, the method used as astarting point was developed on the 6460 Triple QuadrupoleLC/MS (6460).

Collision cell accelerator voltage (CAV) is a key parameter inoptimizing production, detection, and quantitation of ions.Figure 1 illustrates the significant increase in response fordetection of the three steroids (prepared as calibration stan-dards) obtained by changing the accelerator voltage from 5 to2 volts.

Figure 1. Effect on response of changing the cell accelerator voltage (CAV)from 5 volts (lower peaks) to 2 volts (higher peaks).

Figure 2. Significant increase in response due to increasing the multipliervoltage (Delta EMV).

The multiplier voltage (Delta EMV) is another parameter thatsignificantly influences response. By varying the voltage from100 V to 400 V in 100 V increments, the response is increasedby as much as a factor of 14 across the range. In excess of400 volts, the response will still increase, but the signal-to-noise ratio can stay static or indeed decrease. The optimizedvalue was found to be around 200–300 volts (Figure 2).

×103

7.06.86.66.4

Estradiol

400 V EMV

300 V EMV

200 V EMV

100 V EMV

0 V EMV

Estrone

Ethynyl Estradiol

6.26.05.85.65.45.25.04.84.64.44.24.03.83.63.43.23.02.82.62.42.22.01.81.61.41.21.00.80.60.40.2

7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3Acquisition time (min)

8.4 8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.20

Coun

ts

3.0

3.2

3.4

3.6

3.8

4.0

4.2

Estradiol

Cell acc voltage 2 versus 5

Ethynyl estradiol

Estrone4.44.6

×103

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

07.5 7.6 7.7 7.8 7.9 8.0

Acquisition time (min)

Coun

ts

8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9

5

×103

2.0

2.1

1.9

CE voltage Opt. 35–50 V

50 V Opt. 6490

35 V Opt. 6460

1.7

-7.796

1.8

1.6

1.5

1.4

1.3

1.2

1.0

1.1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

7.4 7.5 7.6 7.7 7.8Acquisition time (min)

7.9 8.0 8.1 8.2 8.30

Coun

ts

Figure 3. Optimization of collision energy (CE) on the Agilent 6490 TripleQuadrupole LC/MS System provides an increase in response fordetection of estradiol of almost two fold, compared to the collision energy that was optimal on the 6460 (35 volts).

Optimization of collision energy (CE) is also critical for maxi-mizing response. While the optimal collision energy for the6460 method was 35 volts for estradiol, increasing the voltagein 5 volt increments on the 6490 continually increasedresponse, with a nearly two-fold increase at 50 volts (Figure 3).

One of the keys to the enhanced sensitivity of the Agilent6490 Triple Quadrupole LC/MS System is the proprietaryAgilent iFunnel Technology, which utilizes a novel dual stageion funnel assembly. Both the low and high pressure voltagehave default settings in the tune file of the 6490. However,both voltages can be optimized to significantly increase sensi-tivity. For example, changing the low pressure voltage fromthe default of 60 volts to 100 volts can increase response byas much as 65%, as can changing the high pressure (high RF)voltage from the default of 150 volts to 160 volts (Figure 4).

Applying all of these optimized parameters to the method onthe 6490 results in response that is as much as three timeshigher than that obtained from these steroids using the opti-mized parameters from the 6460 method on the 6490(Figure 5). Applying these optimized parameters enables thedetection of as little as 0.1 pg of estrone (Figure 6), estradiol,and ethynyl estradiol calibration standards on the column (10 pg/L in the sample).

A B2.8

Low pressure ion funnel RF 40–90 volts2.72.6 90 V

70 V80 V

60 V (default)

50 V

40 V

2.52.42.32.22.12.01.91.81.71.61.51.41.31.21.11.00.90.80.70.60.50.40.30.20.1

07.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6

Acquisition time (min)

Coun

ts

8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4

High pressure ion funnel RF

2.72.6

160 V

150 V

2.52.42.32.22.12.01.91.81.71.61.51.41.31.21.11.00.90.80.70.60.50.40.30.20.1

07.3 7.5 7.7 7.9 8.1 8.3 8.5 8.7

Acquisition time (min)

Coun

ts

8.9 9.1 9.3 9.5 9.7

Figure 4. Optimization of both the low pressure and high pressure ion funnel voltages on the Agilent 6490 Triple Quadrupole LC/MS System provides anincrease in response of as much as 65% for both settings, for the three steroids.

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Figure 5. The cumulative effect of the various Agilent 6490 Triple Quadrupole LC/MS System optimization steps onresponse is shown, resulting in as much as much as three times higher response than that obtained from thesesteroids using the optimized parameters from the Agilent 6490 Triple Quadrupole LC/MS System method.

Figure 6. Detection of 1 pg to 100 pg on column of estrone calibration standard, using the optimized method parameters.

0.05

7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5Acquisition time (min)

Coun

ts

8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 9.5

0.15

0.25

0.35

0.45

0.55

0.65

0.75

0.85

0.95

1.05

1.15

1.25

1.35

1.45

1.55

1.65

1.75

1.85

1.95

2.05

2.15

2.25

2.35

2.45×104

Ion funnel optimized

No optimization

CE and CAV optimized

6460 CE, default CAV

08.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 10.0

Acquisition time (min)

Coun

ts

10.2

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4.0

4.4

4.8

5.2

5.6

6.0

6.4

6.8

7.2

7.6

×102

9.8

1 µg/L std = 100 pg on column (1,000 pg/L)

0.1 µg/L std = 10 pg on column (100 pg/L)

0.01 µg/L std = 1 pg on column (10 pg/L)

0.001 µg/L std = 0.1 pg on column (1 pg/L)

Estrone 6490 opt conditions

7

Postcolumn addition of 0.1% ammonia solution at 0.1 mL/minalso gives an increase in response for the steroids whenusing the optimal MS parameters, in the order of 3–4 fold,compared to no addition (Figure 7).

Figure 7. Postcolumn addition of 0.1% ammonia can increase response by as much as almost 4 fold, for all three steroids analyzed.

0.2

7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6Acquisition time (min)

Estrone 1.8 fold increase

Estradiol 3.7 fold increase-8.100

Coun

ts

8.7 8.8 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7

0.4

1.0

1.4

1.8

2.2

2.6

3.0

3.4

3.8

4.2

4.6

5.0

5.4

5.8

6.2

6.6

7.0

×103

7.4

8.9 9.8

Postcolumn addition of 0.1% NH3

No postcolumn addition

Ethynyl estradiol 3.1 fold increase

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Sensitive Detection of Steroids in SewageEffluent.This optimized method can detect sub part per trillion (ng/L)steroids in wastewater. Figure 8 shows ethynyl estradiol at0.91ng/L in crude sewage, as well as the excellent linearity ofthe calibration curve (R2>0.9999). Estrone was easilydetectable at 53.7 ng/L (Figure 9) in crude sewage, and estradiol at 44.5 ng/L (Figure 10). Both of these steroids alsoshowed excellent linearity of quantification, with R2 values>0.9999.

Figure 8. Analysis of 0.91 ng/L of ethynyl estradiol in crude sewage, showing the MRM chromatogram, as well as the chromatogram for theinternal standard and the calibration curve with an R2 value >0.9999.

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Figure 10. Analysis of 44.5 ng/L of estradiol in crude sewage showing the MRM chromatogram, as well as the chromatogram for theinternal standard and the calibration curve with an R2 value >0.9999.

Figure 9. Analysis of 53.7 ng/L of estrone in crude sewage, showing the MRM chromatogram, as well as the chromatogram for theinternal standard and the calibration curve with an R2 value >0.9999.

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Information, descriptions, and specifications in this publication are subject to changewithout notice.

© Agilent Technologies, Inc., 2012Printed in the USAFebruary 24, 20125990-9978EN

Conclusions

The Agilent 6490 Triple Quadrupole LC/MS System withiFunnel Technology provides exquisite sensitivity, making itthe ideal choice for applications that require the detection ofminute quantities of analyte. However, as with any otherinstrument, methods that have been developed on earlier gen-eration MS instruments must be optimized on the 6490 inorder to maximize sensitivity. Such optimization can increasethe response for detection of steroids by as much as threetimes versus the response obtained when using the parame-ters taken from the method developed on the Agilent 6460Triple Quadrupole LC/MS System. The optimized method canprovide sub ng/L detection of steroids in wastewaters, whichare very complex matrices.

References

1. K. A. Kidd, P. J. Blanchfield, K. H. Mills, V. P. Palace, R. E.Evans, J. M. Lazorchak, R. W. Flick, “Collapse of a fishpopulation after exposure to a synthetic estrogen.”, ProcNatl Acad Sci U S A 104, 8897-8901 (2007).

2. Pollutants in Urban Waste Water and Sewage Sludge,http://ec.europa.eu/environment/waste/sludge/pdf/sludge_pollutants_xsum.pdf

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