Commercial Strategies in Non-Target and Suspected Target

Chapter 7

Commercial Strategies in Non-Target and Suspected Target Screening of Water Samples Downloaded by CORNELL UNIV on June 9, 2017 | http://pubs.acs.org Publication Date (Web): December 12, 2016 | doi: 10.1021/bk-2016-1242.ch007

A. B. Sage* and P. Taylor SCIEX, Phoenix House, Lakeside Drive, Warrington, WA1 1RX, United Kingdom *E-mail: [email protected].

The analysis of environmentally related samples (water, soil, air) is of huge importance due to the global impact on human, aquatic and plant life. The effect of known suspect chemical contaminants needs to be thoroughly understood, but of higher concern is the emergence of unknown contaminants caused through bio-transformation. These unknown suspect pose challenges to analytical scientists on how to detect what they are, with the use of LC-MS/MS being used today as a routine frontline technique. In this chapter, we describe the technology and workflows provided by SCIEX which can be used to breakdown those analytical challenges for the analysis of environmental samples using LC-MS/MS.

Introduction Environmental issues, both human and aquatic, are a constant concern due to the fact that billions of grams of chemicals are released into the environment each year (1), ranging from pesticides used in farming and food production, to pharmaceuticals and personal care products flushed into wastewater, to industrial chemical waste of many kinds. The impact of these chemical contaminants on the quality of our air, water and soil, and the health of our plants, our animals, and ourselves is of vital importance to scientists and environmentalists throughout the world. Testing for chemical contaminants in environmental samples is fundamental to understanding their impact, and is thus a growing area of scientific research to allow us to understand the fate, metabolism and impact of such compounds and associated transformation products. There are many tools that © 2016 American Chemical Society

Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Downloaded by CORNELL UNIV on June 9, 2017 | http://pubs.acs.org Publication Date (Web): December 12, 2016 | doi: 10.1021/bk-2016-1242.ch007

analytical scientists can use to profile a whole range of samples, both from a target suspect (known) analysis, through to non-target (unknown) analysis. Instrumental analysis can include techniques such as LC-UV-FLD, IR, NMR, GC & GC-MS, but often these techniques are often plagued with extensive sample preparation protocols, lack of sensitivity, and limitations in the number and classes of compounds targeted per analysis. LC-MS/MS technology, on the other hand, offers the most comprehensive approach for the screening, identification, and quantitation of low-level chemical contaminants in the environment. In this chapter we will describe the LC-MS/MS instrumentation provided by SCIEX for both target and non-target analysis of environmental samples, giving references to typical applications where compounds of interest have analysed using different types of LC-MS/MS instrumentation, as well as highlighting associated software workflows that can help scientists understand complex data sets.

Target Suspect Analysis Using Triple Quadrupole and QTRAP LC-MS/MS Since the introduction of the first commercial triple quadrupole LC-MS/MS instrument in 1989 (API III), SCIEX has been designing and developing LC-MS/MS instruments that have become used globally for many applications. Triple quadrupole mass spectrometry provides a highly selective and sensitive way to detect a specific analyte of interest, and over the last 20 year or so, the sensitivity and speed of such instruments has become significantly better and better allowing more compounds to be detected at lower and lower concentration levels. Triple quadrupole instruments can acquire data in many different ways but are primarily used for target analysis using a scan function called multiple reaction monitoring (MRM). This type of scan function allows a specific analyte to be identified with a high degree of accuracy, reproducibility, selectivity and thus sensitivity. Figure 1 highlights a typical triple quadrupole ion path used with SCIEX instrumentation, whilst Figure 2 shows the current geometry of the SCIEX Triple Quad™ 4500, 5500, and 6500+ platforms where a curved collision cell has been designed to help with instrument geometry and laboratory footprint whilst still maintaining. One of the main key features of the SCIEX LC-MS/MS instrument is around the Ion Source and Ion Introduction technologies – these have been designed over years to be robust and reliable, especially when dealing dirty matrices such as with environmental samples. Turbo V™ ion source provides robust ion production using either electrospray or APCI ionisation techniques, whilst the Curtain Gas Interface and Q0 QJet® technology helps to reduce noise productions whilst keeping the instrument clean. Figure 3 highlights the differences between the original TurboV™ ion source and the new IonDrive™ Turbo V source, where ion production has been increased by using larger and more efficient heating elements to dry and decluster solvent droplets to allows analytes of interest (in ionised forms) to be detected in the mass analyser. Figure 4 highlights a typical MRM approach for target analysis where an analyte of interest is detected by monitoring the precursor and product ion of an analyte that has undergone collision induced dissociation (CID) within the collision cell. 132



Figure 1. Schematic of a Typical Triple Quadrupole instrument. (courtesy of SCIEX)

Figure 2. Schematic arrangement of SCIEX 4500, 5500 & 6500+ Platforms with Curved Collision Cell Design. (courtesy of SCIEX) 133 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 3. Original Turbo V Ion Source (left) with New IonDrive Turbo V (right) designs highlighting large ‘sweet spot’ for increased ion generation. (courtesy of SCIEX)

Figure 4. Schematic of MRM Detection using Triple Quadrupole LC-MS/MS. (courtesy of SCIEX)

To increase data confidence above what you would get with a triple quadrupole LC-MS/MS instrument, SCIEX has unique technology that is called QTRAP®. In the QTRAP® instrument, the third quadrupole mass analyser is replaced by a Linear Ion Trap (LIT). By using the LIT, additional MS experiments can be acquired to compliment above what you would get from a triple quadrupole instrument alone. One such technique is called Information Dependent Acquisition (IDA) where a Product Ion Scan can be triggered for a particular analyte of interest to give a library searchable MSMS spectrum which can be used to confirm the presence of a particular molecule. This type of scan function can also be used in research purposes when looking at non-target 134 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


compounds especially when trying to use MSMS data interpretation to elucidate a compound structure. Another scan function the QTRAP® technology can provide is called an Enhanced Product Ion scan (EPI). The EPI can be acquired in the same instance as acquiring a simultaneous MRM trace. The EPI spectra produced are more sensitive due to the fact that the LIT is used to accumulate MSMS ions and gives a clear ID for a particular ion of interest. The combination of both an MRM and EPI for a particular compound can give the analyst more confidence in the identification by using both ion ratios and full scan MSMS spectra. Figure 5 highlights the schematic of a QTRAP® instrument where the Q3 is replaced by a LIT. Figure 6 highlights the combination of MRM and EPI during a single acquisition and Figure 7 shows the sensitivity gain by doing an EPI over a traditional product ion scan, whilst Figure 8 shows the data. Particular applications where targeted analysis using triple quadrupole or QTRAP LC-MS/MS has been utilized is for the analysis of hormones in water (1) where high sensitivity instruments are used to reach the detection limits required to meet the EPA guidelines, and for the protection of municipal drinking and wastewater sources from contaminants, in this case illegal substances (2), where the presence of the moleculaes have been detected using MRM acquisition but also confirmed using the EPI scan function of the QTRAP in combination with library searching software routines (see Figure 8 as an example). To improve sample throughput and reduce the need to perform offline sample clean-up or pre-treatment, automated LC column switching routines can be employed in the analytical setup whereby two columsn can be used to ‘trap and elute’ the analytes of interest to maintain the robustness of the assay. An example of such is the analysis of acid herbicides and phenyl ureas by LC-MS/MS using large volume injection and automated column switching (3), in relation to municipal water protection. Sample throughput for target analysis is of analytical concern due to the increasing numbers of samples being required to be analysed, but the diversity of chemical compunds being required to be detected also requires more sophisticated methodologies to be employed. An example of this is the use of both simultaneous reversed phase and HILIC chromatography in combination QTRAP LC-MS/MS for the analysis of highly polar, polar and non-polar compounds in wastewater samples (4). In this particular example, both types of chromatographic methods were couple in series to allow a braod diversity of compounds to be analysed in a single method.

Figure 5. Schematic of QTRAP showing where Q3 is a Linear Ion Trap. (courtesy of SCIEX) 135 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 6. Combining MRM and EPI Scans During a Single Analysis and how the MSMS Spectra Produced can be Library Searched. (courtesy of SCIEX)

Figure 7. Highlighting the Sensitivity Gain with EPI Scan on QTRAP. (courtesy of SCIEX) 136 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 8. Utilising MRM-EPI Experiment on QTRAP to Aid Compound Identification and Interpretation. (courtesy of SCIEX)

Target Suspect and Non-Target Analysis Using High Resolution Accurate Mass (HRAM) LC-MS/MS To compliment a targeted analytical approach using SCIEX Triple Quad™ or QTRAP® instrumentation, which are generally regarded as low resolution/nominal mass analysers, the development of high resolution time-of-flight (TOF) and Quadrupole Time-of-Flight (QTOF) instruments has gained usage also over the last 20 years since commercialisation of such technology. Similar to quadrupole instrumentation, QTOF technology has also developed through advances in science to allow instruments to provide high spectral resolution (>30,000 FWHM), high accuracy measurement (sub 1ppm) with high dynamic range (4 orders) and high sensitivity (sub ppb) detection. These high resolution instruments have also been developed with highly sophisticated software workflows that allow a relatively unskilled analyst to collect extremely specific data with ease. The QTOF instruments today can also be used in a targeted way to look for compounds that we know are in our sample, but because they acquire ‘full scan’ information all of the time, they can also provide information on what we ‘don’t’ know is in our sample – as an analyst, you just need to go and look to see what you can see. This non-targeted (unknown) approach can help scientist understand the fate of particular molecules in the environment, be able to understand what type of transformations occur under certain conditions, and thus help guide remedial procedures to help with protecting both aquatic and human life from potential pollution or bio-accumulation. 137



High resolution accurate mass instruments, such as a QTOF provides a typical performance that a scientist can use to identify a known target with high accuracy (and thus confidence) but also allow a compound that is new to be ‘identified’ through the empirical calculation of an elemental formula and also its structure via accurate mass MSMS interpretation. SCIEX has a range of high resolution LC-MS/MS instrument over two distinct but complimentary platforms. The SCIEX TripleTOF® instruments, which comprise of a 5600+ and 6600 product, are research grade high resolution instruments with flexible sample introduction solutions to do standard flow HPLC/UHPLC, nano scale LC or more recently Microflow LC. The X500R QTOF instrument, introduced in 2015, has been designed to be a more compact design whilst maintaining high resolution accurate mass performance to deliver 35K FWHM resolution and sub 1ppm accuracy of mass measurement, and is primarily designed to be used with standard flow HPLC/UHPLC for more routine target and non-target applications. Both platforms have the key attributes as previously described with the SCIEX Triple Quad™ instruments (Turbo V™ Ion Source, Curtain Gas, QJet® ion sampling, LINAC collision technology), but with unique Time of Flight mass analysers that can acquire data at very high speed in both MS and MSMS mode to ensure that a sample can be analysed in a comprehensive way as possible. The X500R QTOF, which has been designed with new technology built into the analyser, maintains a high resolution analysis (typically 35K FWHM) whilst maintaining sensitivity (sub ppb) using what is called N-Optics within the flight tube. Traditional and older generation TOF instruments tended to trade resolution with sensitivity due to the loss of transmission with longer flight tubes and inefficient ion sampling optics. With X500R, both resolution and sensivity is maximised to give the best result possible. But with every high resolution analysis, the end result is to give a total digital record of a sample that can be interrogated over and over again, depending what question the analyst is asking. Figure 9 shows a picture and schematic of the SCIEX 5600+ TripleTOF® and its associated TOF analyser, whilst Figure 10 shows the X500R and the schematic of the N-Optic designed TOF analyser. When used in a targeted way, the newer generation of high resolution QTOF LC-MS/MS instruments can perform similar to a triple quad instrument in terms of sensitivity and dynamic range, but with the added benefit of highly accurately measured MS and MSMS data. Two examples on the use of high resolution QTOF is for the targeted quantitation and additional identification of pharmaceuticals and personal care products (PPCPs) in environmental and water samples (5, 6). These examples highlight the current quantative performance of QTOF instruments in combination of how accurate mass measured MS and MSMS data can be used to additionally confirm the presence of target molecules via library searchable accurate mass measured spectra. In combination with the hardware, software algorithms and solutions are used to provide both quantitative analysis (MultiQuant™), non-target analysis (MasterView™ with library view and Peakview®) to aid identification of compounds within a sample, and MarkerView™ for doing PCA and statistical analysis in relation to metabolic or environmental fate studies. These packages provided by SCIEX can process data efficiently and quickly, but also compliment 138



the use of external database such as Chemspider for the identification of structural MSMS information in relation to compound ID. This type of workflow is important when non-target (suspect) compaounds have been identified in a particular sample and can help the analyst understand more about what type of molecule is present. Figure 11 highlights the overall workflow for both target and non-target analysis using high resolution LC-MS/MS instrument, along with software processing. In the targeted approach, we can look for analytes that we suspect are in the samples by directing the instrument to collect MS/MS spectra (for example in a data dependent way such as in an MRM) and then quantitate the analyte of interest. Here we use MultiQuant software to determine the concentration levels of the compounds we know are in the sample. In an non-targeted application, we can also look for the compounds that we suspect are there using a data dependent acquisition (in this case Information Dependent Acquisition or IDA), where we again collect high resolution MS/MS spectra for each of the compounds and confirm their presence with ion ratios and library searching using PeakView and Masterview. At the same time, we can also use the IDA approach to collect MS/MS spectra on ions that are presented to the detector above a certain threshold, and thus either compare these spectra to the MS/MS library via MasterView or if we don’t find a match in the library, send the MS/MS to a database such as Chemspider where we use the MS/MS information to try to elucidate the structure of the potential molecule. Here we use the accurate mass measurements from the MS/MS spectra, the relevant ion ratios and also the isotopic patterns of the pseudo-molecular ions/adducts. By doing this, we provide the confidence to the analyst that we have identified what we expected in the sample, but at the same time we have collected MS/MS information on as many suspect molecules within the sample to ascertain their potential structure. This overall for non-target and statistical analysis is highly in two articles showing how the importance of software in an overall analytical workflow helps with the compound identification but also with understanding the fate of these molecules within the environment – an importance for the protection of both human, plant and aquatic health and a concern for the future of the environment of our planet (7, 8).

Figure 9. SCIEX TripleTOF® 5600+ and TOF Analyser. (courtesy of SCIEX) 139 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 10. X500R QTOF and N-Optic Design TOF Analyser. (courtesy of SCIEX)

Figure 11. Target and Non-Target Workflow using SCIEX High Resolution LC-MS/MS instrumentation and Software. (courtesy of SCIEX)

140 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Sanchez L.; Yoo, L.; Noestheden, M. EPA Method 539: Hormones in Drinking Water Using the QTRAP® 6500 LC/MS/MS System; 1 Orange County Water District, Fountain Valley, California (USA); 2 SCIEX Concord ON, Canada. Publication number: 15-01, 2015; www.sciex.com. Berset, J. D.; Scherer, M; Schreiber, A. Quantitation and Identification of Legal and Illicit Drugs in Wastewater in the low Nanogram per Liter Range using Large Volume Direct Injection and QTRAP® Technology; 1 Water and Soil Protection Laboratory (WSPL), Office of Water and Waste Management, Bern, Switzerland, 2 SCIEX Brugg, Switzerland, 3 SCIEX Concord, ON, Canada. Publication number: 11130615-01, 2015; www.sciex.com. Thomas, J.; Struthers, S.; Lock, S. The Detection of Acidic Herbicides and Phenyl Ureas by LCMS/MS with Large Volume Injection and Automated Column Switching; 1 SEPA, East Kilbride, UK, 2 SCIEX Warrington, U.K. Publication number: 3370611-01, 2011; www.sciex.com. Boltner, A.; Schröder, W.; Grosse, S.; Letzel, T.; Schreiber, A. Simultaneous Characterization of Highly Polar, Polar and Nonpolar Compounds in Wastewater using Serial Coupled RPLC and HILIC with a QTRAP® 5500 LC-MS/MS; 1 Technical University of Munich, Chair of Urban Water Systems and Engineering, Garching, Germany. 2 SCIEX Concord, ON, Canada. Publication number: RUO-MKT-02-3765-A, 2016; www.sciex.com. Latawiec, A.; Schreiber, A. Analysis of Personal Care Products (PPCP) in Water Samples by Way of Large Volume Sample Injections; SCIEX, Concord ON, Canada. Publication number: 10160814-01, 2014; www.sciex.com. Schreiber, A. Quantitation and Identification of Pharmaceuticals and Personal Care Products (PPCP) in Environmental Samples using Advanced TripleTOF® MS/MS Technology; SCIEX, Concord ON, Canada. Publication number: 7200213-02, 2013; www.sciex.com. Schreiber, A. MultiQuant™ Software Version 3.0- Improving Data Quality and Processing Throughput with Better Peak Integration, Quantitative and Qualitative Compound Review for the Analysis of Food, Drinking Water, and Environmental Samples; SCIEX, Concord ON, Canada. Publication number: 8160213-01, 2013; www.sciex.com. Schreiber, A.; Cox, D.; Pace, N.; Borton, C. Metabolomic Profiling of Accurate Mass LC-MS/MS Data to Identify Unexpected Environmental Pollutants; SCIEX, Concord ON, Canada. Publication number: 0460210-01, 2010; www.sciex.com.

141 Drewes and Letzel; Assessing Transformation Products of Chemicals by Non-Target and Suspect Screening Strategies and ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Commercial Strategies in Non-Target and Suspected Target

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