analysis of alkylphenols using gc-ms/ms and automated srm … · 2018. 3. 20. · and nonylphenol...

Analysis of alkylphenols using GC-MS/MS and automated SRM development

AuthorsInge de Dobbeleer, Joachim Gummersbach, Hans-Joachim Huebschmann; Thermo Fisher Scientific, Dreieich, Germany

Keywords Chromeleon Chromatography Data System, Automated build of quantitation method, Environment, Isomer integration, Linearity, Repeatability

Goal To describe the analysis of alkylphenol compounds in food and environmental matrices, illustrating the productivity and high-quality results from the GC-MS/MS system with automated selected reaction monitoring (SRM) development.

APPLICATION NOTE 10407

IntroductionAlkylphenols and ethoxylated alkylphenols are used as surfactants in a broad range of applications, from processing wood and metals, to use as emulsifiers in polymerization, and even as solvents for flavorings.

Alkylphenols are endocrine disruptors with estrogen-like properties that have been shown to alter mammary gland development.1,2 Additionally, both ethoxylated alkylphenols and alkylphenols are toxic to aquatic life. A subfamily of these chemicals, the nonylphenols, were once widely used in household laundry detergents. Although nonylphenol ethoxylate has been banned for use in household laundry detergents in both the E.U. and the U.S., it still widely used in industrial laundry detergents and is particularly persistent in water.3

Due to their toxic qualities, production and use of nonylphenol and nonyphenol ethoxylates is prohibited in the E.U., and these chemicals are included in the list of hazardous compounds in the European Water Framework Directive.4,5

2

Current European regulations create high demand for analytical screening for these compounds at low levels in both water and food. Additionally, nonylphenol and nonylphenol ethoxylate have been part of a U.S. Environmental Protection Agency (EPA) action plan since 2010.3

Experimental conditionsSample preparationWater samples

To 1 L of sample; n-hexane was added and the mixture was shaken. After separation of the water and organic phases, the organic phase was removed and dried with anhydrous Na2SO4. An aliquot of the organic extract was evaporated to a volume of 3–4 mL and then evaporated under a gentle nitrogen stream to the final volume.

Solid samples

Into a glass jar, 10 g of the solid sample was weighed, then anhydrous Na2SO4 and 40 mL of extraction solvent mixture (hexane and acetone) were added. The glass jar was sealed with a Teflon® seal and sonicated for 20 min. An aliquot of the sample extract was placed into a Kuderna-Danish apparatus. Another 40 mL of extraction solvent mixture was added to the sample and the extraction was repeated. An aliquot of the second extraction was added to the first extraction aliquot. The combined extracts were evaporated to a volume of 3–4 mL and then evaporated under a gentle nitrogen stream to the final volume.

Method setupMeasurements were carried out using the Thermo Scientific™ TRACE™ 1310 gas chromatograph coupled to the Thermo Scientific™ TSQ™ Duo* triple quadrupole mass spectrometer. The TRACE 1310 GC system was equipped with a programmed temperature vaporizing (PTV) multimode injection system to achieve optimum sample transfer into the analytical column for the critical alkylphenol compounds. The analytical conditions of the GC-MS/MS method are provided in Table 1.

The selected reaction monitoring (SRM) mode of the TSQ Duo* GC-MS/MS system was chosen to achieve the lowest possible detection limits with high compound selectivity for the typical matrix-laden extracts from food or environmental samples.

The Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) software was used as the data system for sample analysis and quantitative data processing. The unique functionality of the Chromeleon CDS software is critical to the integration of the compound isomers into a single final result.

AutoSRM was used for the analytical method development. The automated SRM function is an important aid in optimizing the SRM transitions on all levels of method development. The precursor ion, product ion choice, and collision energy are optimized in a few logical, quick, and easy-to-follow steps. The result is a ready-to-use instrument and quantitation method.

Figure 1. TSQ Duo GC-MS/MS system with Thermo Scientific™ TriPlus RSH™ Autosampler.

* Equivalent or better performance with the Thermo Scientific™ TSQ™ 9000 GC-MS/MS system

3

Table 1. Recommended instrument conditions.

TRACE 1310 GC

Injection volume: 1 µL

Inlet liner: Siltec baffled liner (P/N 453T2120)

Carrier gas: He, constant flow, 1.0 mL/min

Column type: TG-5MS, 20 m length × 0.18 mm ID × 0.18 µm film (P/N 26098-5780)

Oven program: 75 °C, 2.2 min

30.0 °C/min to 200 °C

20.0 °C/min to 300 °C

300 °C, 1.0 min

Transfer line 320 °C Temp.:

PTV Injector

Injection mode: Splitless with surge pressure

Surge pressure: 345 kPa

Surge time: 1 min

Split time, flow: 2 min, 30 mL/min

Inject temp.: 60 °C, 0.1 min

Transfer temp.: 600 °C/min to 280 °C, 5 min

Cleaning temp.: 870 °C/min to 325 °C, 15 min

Clean split flow: 25 mL/min

TSQ Duo* Mass Spectrometer

Source temp.: 280 °C

Ionization: EI, 70 eV

Emission current: 50 µA

Mass resolution: Q1, Q3 normal (0.7 Da at FWHM)

Collision gas: Argon

Compounds and transitionsThe complete list of compounds and transitions as prepared by the AutoSRM tool is shown in Table 2.

All analytical information with retention times and SRM transitions is stored in the Chromeleon CDS method database. This information is also used for the quantitation processing method comprising a method including all compounds, their transitions, and retention times. This information storage strategy avoids potential human error in writing or copying from one file to another and simplifies the complete method setup.

Results and discussionThe alkylphenol compound class includes many isomers, which do not elute as a single peak but as a group. These isomers require special attention for reliable signal integration and representation of the complete isomer peak area of the compound. The Chromeleon CDS software offers the ability to define a retention time range for the integration of these isomeric compounds, as provided in Table 3. Quantitation is performed using the elution time range for the complete isomer group.

In Figure 2, the complete MRM chromatogram is shown for a mixture of alkylphenols listed in Table 1.

Automated SRM method developmentManual SRM method development can be labor intensive and time consuming. For every compound, one precursor ion, but preferably two or more precursor ions, need to be selected. Subsequently, the optimum collision energy and product ions for detection need to be defined for each precursor ion. When investigating dozens or hundreds of compounds, it is challenging for any analyst to perform this task.

In addition to all the necessary injections and data gathering, the primary challenge lies in handling the data and carefully monitoring which precursor yields the best product ion at which collision energy at a certain retention time, and performing this at the same time for dozens of compounds.

The automated SRM feature of the Chromeleon CDS software was designed to perform these tasks automatically using the following workflow:

1. A full-scan experiment identifies the target compounds.

2. The most intensive or selective ions are selected and put into a working list.

3. A product scan experiment is run in the next injection using the working list.

4. The method and the sequence for the product scan experiments is prepared automatically.

5. The product scan spectra are clearly shown by compound and by precursor ion.

6. The most intensive or selective product ions are selected and put into the working list.

7. Collision energy is optimized for sensitivity.

*Equivalent or better performance with the TSQ 9000 GC-MS/MS system

4

8. The method and the sequence for the optimal collision energies is set up automatically.

9. The completed working list is stored as a compound database csv-file. This file can be imported or attached to the instrument method and is also used as the quantifying processing method.

The sequence of the AutoSRM steps is illustrated in Figures 4–5.

The Chromeleon CDS software can perform library searches for compound identification. Optionally, Chromeleon CDS software can import a list of compounds with retention times, etc. All of the data can be reviewed and edited manually.

Quantitative calibrationThe calibration curves (Figure 6) show good linearity over a range of 2 to 200 ppb. Even at the lowest level, the peaks are very clearly present, even for the isomer groups and the confirming ions.

Repeatability in matrixA spiked water sample was extracted and injected seven times. Table 4 shows repeatability of results at the relevant low concentration levels. The repeatability in a water matrix is excellent; all compounds show a repeatability below 5%.

Trend reportingThe Chromeleon CDS software offers the ability to report trends and make flowcharts (Figure 7).

Parent Mass [m/z] Product Mass [m/z] RT [min] Name179 45 8.69 4-nonylphenol

179 73 8.69 4-nonylphenol, confirming ion 1

246 105 11.65 4-nonylphenol diethoxylate (SS2)

246 133 11.65 4-nonylphenol diethoxylate (SS2), confirming ion 1

339 73 11.67 4-octylphenol triethoxylate

339 117 11.67 4-octylphenol triethoxylate, confirming ion 1

339 161 11.67 4-octylphenol triethoxylatee, confirming ion 2

357 73 10.25 BisphenolA

357 191 10.25 BisphenolA, confirming ion 1

185 45 8.66 D8-4-nonylphenol (SS1)

185 73 8.66 D8-4-nonylphenol (SS1), confirming ion 1

207 45 7.75 nonylphenol

207 73 7.75 nonylphenol, confirming ion 1

251 73 9.57 nonylphenol monoethoxylate

251 135 9.57 nonylphenol monoethoxylate, confirming ion 1

251 207 9.57 nonylphenol monoethoxylate, confirming ion 2

179 73 8.1 4-n-octylphenol, confirming ion 1

179 45 8.1 4-n-octylphenol

295 73 10.43 4-t-octylphenol diethoxylate

295 207 10.43 4-t-octylphenol diethoxylate, confirming ion 1



339 117 12.15 nonylphenol triethoxylate, confirming ion 1

339 73 12.15 nonylphenol triethoxylate

339 161 12.15 nonylphenol triethoxylate, confirming ion 2

207 73 7.04 4-t-octylphenol

207 45 7.04 4-t-octylphenol, confirming ion 1

295 207 11.4 nonylphenol diethoxylate, confirming ion 1

295 117 11.4 nonylphenol diethoxylate

295 135 11.4 nonylphenol diethoxylate, confirming ion 2

295 73 11.4 phenolnonylphenol diethoxylate, confirming ion 3

Table 2. List of compounds in the method automatically prepared by AutoSRM.

5

Table 3. Isomer compound integration time ranges.

Figure 2. Chromatogram of alkylphenol standard at 100 ng/mL.

Figure 3. Chromatogram showing the integrated range of nonylphenol isomers. Top: transition 207 > 73 Bottom: transition 207 > 45

6.55 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 12.85-1.0e7

-5.0e6

0.0e0

5.0e6

1.0e7

1.5e7

2.0e7

2.5e7

3.0e7

3.5e7

4.0e7

4.5e7

5.0e7

5.5e7

6.0e7

6.5e7

7.0e7

7.5e7

8.0e7

min

countsAlkylphenols_Water_SRM #5 AEO5 TIC

7.320 7.400 7.500 7.600 7.700 7.800 7.900 8.000 8.120-1.0e6

0.0e0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e67.84;287661.;nonylfenolGroupStart = Fixed GroupEnd = Fixed

min

counts

Con

f 1

1 - Alkylphenols_Water_SRM #5 Ion = 45-2.0e60.0e0

2.5e6

5.0e6

7.5e6

1.0e7

1.3e7

1.5e7

1.8e77.84;787922.;nonylfenol

GroupStart = Fixed GroupEnd = Fixed

min

counts

Qua

n

2 - Alkylphenols_Water_SRM #5 Ion = 73

Target Compound RT [min]

4-t-octylphenol 7.02nonylphenol 7.5-84-n-octylphenol 8.07D8-4-nonylphenol 8.644-nonylphenol 8.67nonylphenol monoethoxylate 9.35–9.8bisphenol A 10.234-t-octylphenol diethoxylate 10.41nonylphenol diethoxylate 10.8-124-nonylphenol diethoxylate 11.634-octylphenol triethoxylate 11.65nonylphenol triethoxylate 11.9–12.4

6

Figure 4. Chromeleon AutoSRM screen for automated GC-MS/MS method development. Upper left: The list of compounds and the retention times

Upper right: The list of most intense ions in the working list Lower left: The full-scan chromatogram Lower right: The full-scan spectrum of a selected compound

Figure 5. Optimization of the SRM collision energy by AutoSRM. Upper left: The list of compounds and the retention times

Upper right: The list of applied collision energies and sorted by intensity of the product ion in the working list Lower left: The SRM peak, in this case m/z 185 > 73 Lower right: The graph of collision energy versus intensity

7

0 20 40 60 80 100 120 140 160 180 200 220 240 2500

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000counts*min4-nonylfenol @MT_4-nonylfenol_Quan

0 20 40 60 80 100 120 140 160 180 200 220 2402500

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000counts*min4-t-Octylfenol diethoxylat @MT_4-t-Octylfenol diethoxylat_Quan

0 125 250 375 500 625 750 875 1000 1125 12000

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000counts*minnonylfenol triethoxylat @MT_nonylfenol triethoxylat_Quan

0 125 250 375 500 625 750 875 1000 1125 12000

25000

50000

75000

100000

125000

150000

175000

200000

225000

250000

275000

300000

325000

350000

375000

400000

425000

450000counts*minnonylfenol monoethoxylat @MT_nonylfenol monoethoxylat_Quan

10.300 10.350 10.400 10.450 10.500-2,000

0

5,000

10,000

15,000

20,000ICIS

min

counts

Con

f 2

1 - Alkylphenols_Water_SRM #1 135 Ion = 135-5,0000

10,000

20,000

30,000 10.40;429.;4-t-Octylfenol diethoxylatICIS

min

counts

Con

f 1


20,000

40,000

60,000

80,000

100,000ICIS

min

counts

Qua

n

3 - Alkylphenols_Water_SRM #1 73 Ion = 73

10.41;1544.;4-t-Octylfenol diethoxylat

10.41;273.;4-t-Octylfenol diethoxylat

11.800 11.900 12.000 12.100 12.200 12.300 12.400-2,000

10,00014,000

12.19;1037.;nonylfenol triethoxylatGroupStart = Fixed

min

counts

Con

f 2


12,500

25,00030,000

12.18;2421.;nonylfenol triethoxylatGroupStart = Fixed

min

counts

Con

f 1


10,00014,000

12.19;1025.;nonylfenol triethoxylatGroupStart = Auto

min

counts

Qua

n


8.550 8.600 8.650 8.700 8.750-50,000

0

100,000

200,000

300,000

400,000

500,000ICIS

min

counts

Con

f 1

1 - Alkylphenols_Water_SRM #1 45 Ion = 45-2.0e50.0e0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6ICIS

min

counts

Qua

n


8.66;6732.;4-nonylfenol

8.66;18757.;4-nonylfenol

9.200 9.400 9.600 9.800 10.000-10,000

0

25,000

50,000

75,000

100,000110,000

9.66;3893.;nonylfenol monoethoxylat


min

counts

Con

f 2


0

12,500

25,000

37,500

50,00055,000

9.66;2243.;nonylfenol monoethoxylat


min

counts

Con

f 1


0

25,000

50,000

75,000

100,000

120,0009.66;4858.;nonylfenol monoethoxylat

GroupStart = Fixed GroupStart = Fixed

min

counts

Qua

n


Figure 6. Calibration curves and peaks at lowest level.

Calibration Curve Compound

4-nonylphenol R2 = 0.9973 Peak at 2 ng/mL

Nonylphenol monoethoxylate R2 = 0.9997 Peak at 10 ng/mL

4-t-octylphenol diethoxylate R2 = 0.9989 Peak at 2 ng/mL

Nonyl phenol trietoxylate R2 = 0.9975 Peak at 10 ng/mL

Conclusions• All alkylphenol compounds show good linear calibrations

over the range of 2 to 200 ppb.

• The method has proven high precision and repeatability in a water matrix sample.

• AutoSRM is a useful tool for automated MRM method development and maintenance. It delivers an optimized method and avoids tedious and laborious method development.

• The Chromeleon CDS software offers excellent processing options for data reviewing, including ion ratio checks and reporting. All reporting can be optimized for each application requirement.

References1. Chain of Contamination – The Food Link; Worldwide Fund for Nature (WWF) 2006.

2. Directive 2003/53/EC of the European Parliament and of the Council of 18 June 2003 amending for the 26th time Council Directive 76/769/EEC relating to restrictions on the marketing and use of certain dangerous substances and preparations (nonylphenol, nonylphenol ethoxylate and cement). Off. J. Eur. Communities: Legis., 2003.

3. Nonylphenol (NP) and Nonylphenol Ethoxylates (NPEs) Action plan [RIN 2070-ZA09]; U.S. Environmental Protection Agency, Washington, DC, 2010.

4. Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy. Off. J. Eur. Communities: Legis., 2001.

5. Application Brief 52298, Introducing AutoSRM-MRM Simplicity for High Performance Results, Thermo Scientific, Austin, TX, 2012.

Find out more at thermofisher.com/unstoppableGCMS

©2018 Thermo Fisher Scientific Inc. All rights reserved. Teflon is a registered trademark of E.I. du Pont de Nemours. All other trademarks are the property of Thermo Fisher Scientific. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. AN10407-EN 0318M

Table 4. Method precision in % RSD measured, n = 7.

Compound Avg. Conc. [ppb]

% RSD

4-n-octylphenol 81.9 1.494-nonylphenol 79.2 1.674-nonylphenol diethoxylate 731 4.254-octylphenol triethoxylate 86.4 3.594-t-octylphenol 95.7 1.234-t-octylphenol diethoxylate 97.9 2.1bisphenol A 79.2 4.07D8-4-nonylphenol 324.5 1.81nonylphenol 370.3 1.55nonylphenol diethoxylate 464.6 3.87nonylphenol monoethoxylate 466 1.82nonylphenol triethoxylate 513 4.23

Figure 7. An example of the area of the internal standard in a short sequence, showing the average and the 2s and 3s values as horizontal bars. The analytical data of this sequence is well within the 2s width.

http://thermofisher.com/unstoppableGCMS

analysis of alkylphenols using gc-ms/ms and automated srm … · 2018. 3. 20. · and nonylphenol...

Documents