Target, Suspected-Target, and Non-Target LC-MS ... - ACS Publications

Chapter 8

Downloaded by UNIV OF NEW ENGLAND on February 23, 2017 | http://pubs.acs.org Publication Date (Web): December 12, 2016 | doi: 10.1021/bk-2016-1242.ch008

Target, Suspected-Target, and Non-Target LC-MS(/MS) Screening: New (Practical) Strategies for CECs in Water Bodies Olaf Scheibner,*,1 Anthony Squibb,2 Giorgia Greco,2 and Frank Steiner2 1Thermo 2Thermo

Fisher Scientific, Im Steingrund 4-6, 63303 Dreieich, Germany Fisher Scientific, Dornierstrasse 4, 82110 Germering, Germany *E-mail: [email protected].

Environmental analysis faces new challenges in terms of sensitivity, accuracy and speed of analysis nearly every day. Latest ultra high pressure liquid chromatography technology and ultra high resolution mass spectrometry technology from Thermo Fisher Scientific™ helps scientists to meet every day’s demands in environmental analysis and latest software technology makes the analyses faster and easier to be accomplished. Thermo Scientific™ Vanquish™ liquid chromatography systems and Orbitrap™ high resolution mass spectrometry systems together with Chromeleon™, TraceFinder™ and Compound Discoverer™ software provide integrated workflows for a fast and easy way from sample to result for targeted quantitation, targeted screening and unknown screening tasks.

Introduction Many analytical laboratories all over the world are focusing their work on the study of chemicals of emerging concern (CECs) in water. In this field, a limited number of compounds are currently under government scrutiny due to their possible negative effects on the environment and human health. However, as they enter into the environment these compounds can undergo a series of transformations that are difficult to predict. For this reason, government institutions are directing both academic and public attention to the investigation © 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.


of such chemicals, since they may pose a risk even higher than the original compound. Little information is available on such chemicals and often reference materials are not commercially available for a target analysis. In most cases the type of analysis performed is classified as suspected-target analysis (a specific list of compounds is screened, but no reference compounds are available for confirmation) or non-target analysis (no hypothesis is made as to which compounds are present in the sample; the aim is to analyze everything). In both types of analyses compound identification is based on the use of databases and libraries. As a consequence, the more independent parameters used for the characterization of a compound are available, the higher will be the confidence in the identification.

Figure 1. LC-MS/MS Instrumentation: Thermo Scientific™ Q Exactive™ Focus Mass Spectrometer with Vanquish™ Horizon UHPLC System.

Achieving low limits of detection (LODs) of pesticides, antibiotics and veterinary drug residues in food and drinking water is of paramount importance in order to monitor the regulatory levels as stated by the US, Japanese and EU directives. These substances pose a significant health and environmental threat, and therefore need to be accurately detected at the lowest levels, typically at parts per trillion (ppt). Traditionally, LC-MS/MS has been used by the environmental and food industries for the identification and quantitation of these residues (Figure 1). However, this methodology typically requires extensive offline sample preparation, which can be time consuming and expensive. Within the field of environmental analysis, the demand for quick and simple techniques to analyze large numbers of samples is growing each year. While the limits of quantitation (LOQs) required by government authorities are continuously lowered, the number of analytes of interest is growing exponentially. 144

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.


An online-SPE system that allows for sample injection, pre-concentration, and chromatographic analysis in one step is the key solution here. It reduces sample preparation time from hours to minutes and sample consumption from liters to milliliters. The combination of non-polar and polar pre-concentration columns in one setup even allows the user to analyze a very broad spectrum of analytes ranging from non-polar to very polar. By using high-resolution, accurate mass (HRAM) liquid chromatographymass spectrometry (LC-MS) (at least 50,000 resolution) and full-scan experiments, compound identification, screening and quantitation for an unlimited number of compounds in a targeted or non-targeted screening approach can be accomplished with only one chromatographic run.

Combining Chromatography with Mass Spectrometry The quality of LC-MS analyses depends on the combined capabilities and performance of both the mass spectrometer and the liquid chromatography instrument. The specific chromatographic retention time under defined conditions provides additional information for compound identification. Even more important is the positive impact of the compound separation prior to the mass spectrometer ion source. Efficient LC separations account for electrospray ionization with lowest possible interference, namely less matrix suppression, allowing accurate compound detection at lower LODs. State of the art UHPLC technology helps significantly in obtaining excellent analytical results from advanced mass spectrometry, in particular for ultra-trace analysis in complex samples or those with harsh matrices. The chromatographic resolution is controlled by a combination of the column and the instrument performance while the precision of chromatographic retention primarily depends on the instrument. Hence the UHPLC instrument provides the enabling technology to obtain the best possible performance from a given column.

UHPLC for Separation of Complex Samples The goal to achieve the optimized separation efficiency in the shortest time possible requires smaller stationary phase particles (typically sub-2 µm) applied at higher linear mobile phase velocities. These are packed in small diameter columns (typically 2.1 mm) in order to keep the flow rates at even elevated linear velocities below 1 mL/min for better compatibility with the ESI-MS. While the small particles enable separation of samples with low complexity on very short column lengths, it is the combination of small particles packed in longer columns up to 250 mm or even the application of serially coupled columns that helps to resolve highly complex samples. This approach is relatively tedious, as resolution only increases with the square root of the column length. In any case it requires instruments that enable higher operating pressures and reduced system volumes. Indeed, if the system extra column volume is too large for the column in use, the band dispersion outside the column reduces the chromatographic resolution significantly. Superficially porous particles achieve impressive separation 145


efficiency even with particle sizes between 2 and 3 µm which reduces the ratio of operating pressure to performance substantially. This often gave rise to the false assumption that the respective columns could also be successfully operated in conventional HPLC systems. In fact the best chromatographic results are enabled by the performance of a UHPLC system and depend on a number of parameters: 1. 2.


3. 4. 5.

6.

The combination of flow and pressure specifications The accuracy and precision of gradient formation and the gradient dwell volume The accuracy, precision and the absence of pulsation during flow generation The precision of small volume sample injection with lowest sample carryover The accuracy and stability of column and mobile phase temperature control and how the column thermostat handles the generation of frictional heat under elevated column pressure The lowest possible extra column band dispersion and how dead-volume free connection of the column to the instrument is facilitated

The Thermo Scientific™ Vanquish™ Horizon UHPLC system marks the latest UHPLC technology development from Thermo Fisher Scientific and was designed to incorporate all the requirements listed above (1, 2). With a maximum operating pressure of 1500 bar (22,000 psi) it facilitates the use of small particles with both short and long columns. All fluidic connections in the Vanquish system are based on Thermo Scientific Viper™ capillaries. Viper is a tip-sealing fitting concept and works with a seal ring directly at the fluidic contact between the capillary and its counterpart (Figure 2). Relative to ferrule-based systems this provides the massive advantage of achieving virtually zero dead volumes as soon as the fluidic connection is tight. It is tool-free and can be safely hand-tightened for pressures up to 1500 bar (22,000 psi), which is an essential pre-requisite for successful operation in the Vanquish system. It can markedly improve all fluidic connections which will translate into narrower and more symmetrical peaks as shown in Figure 2. A further advantage of Viper is the small design of the end-fittings with the removable knurl that makes it easy to use when space is limited and allows for plumbing to any 10-port switching valve.

Improved Retention Time Precision for Most Accurate Retention Index Calculation The analysis of chemicals of emerging concern in water bodies is predominantly performed by LC-MS methods. However, compound identification is based only on mass spectrometric data (high resolution MS, isotopic pattern, MS2 data). It should not be forgotten that LC can deliver a very important parameter for the identification: the retention time. Why is retention time so underestimated? The retention time, is used as an identification criterion in many laboratories, but it is not yet used, unlike MS data, in databases or in 146 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.


inter-laboratory studies. This is because the diversity of LC conditions in terms of eluent, gradient, column dimension and chemistry, to mention but a few, can make the comparison among laboratories almost impossible. The possibility to share this information among academic and contract laboratories as well with governmental institutions is of utmost value.

Figure 2. a) Technical details of a Thermo Scientific Viper™ capillary. b) Exemplary compound analyzed with an LC system with Viper™ capillaries or PEEK capillaries. Viper™ capillaries provide better peak shape and efficiency. A valid approach to overcome this difficulty is the use of the Retention Time Index (RTI) (3). The RTI is a model to standardize LC retention time values, following the same concept of the Retention Index (RI) well known in the field of GC-MS. Without going into the details of the method (for a more in depth discussion, please refer to ref. (3)), the calculation of the RTI is based on the analysis with your specific LC method of a mixture of 10 reference standards with increasing polarity. An RTI value of 50 is assigned to the first eluting compound, and RTI of 150 to the last one. The other reference standards are normalized on the basis of their polarity, expressed as logarithm of the partition or the distribution coefficient (log P or log D). The retention time of each suspected or unknown compound is afterwards normalized to RTI using the straight line functions that connect two consecutive RTI values. RTI values are independent form the specific LC conditions and can therefore be used in databases and for inter-laboratory comparison to increase the confidence in compound identification.

How Does the Retention Time Precision Affect the RTI Value? RTI values can be shared among laboratories and accurate RTI values are fundamental for compound identification. In the calculation of the RTI the precision of the retention times of the reference standards and of the suspected/unknown compounds have a direct effect on the RTI error. Let’s consider two reference standards with retention time at 14 min and 15 min, and an unknown compound that elutes at 14.5 min. Let’s assume that the relative standard deviation on the retention times is equal to 0.5% (a reasonable value observed in many laboratories). This error in the retention times will result in an error on the RTI equal to 1, which is a quite significant effect on the accuracy of the RTI. However, LC systems that can deliver outstanding retention time precision run after run and day after day with relative standard deviations on 147



the retention times equal to or lower than 0.01%, will give highly accurate RTI values, with negligible standard deviation of 0.02. This excellent LC performance can be achieved by cutting edge technology, as offered by the Vanquish™ Horizon UHPLC System. In particular, the advanced parallel piston design with independent linear drives and adaptive thermal effect compensation for pulsation free flow delivery account for excellent gradient and flow control with a mixing volume as low as 35 µL (1). Combined with the SmartInject concept as an intelligent pre-compression of the sample liquid in the injector loop, this enables continuous flow delivery for excellent retention time precision and also extends column life time, as the pressure dip at the switching point of the injection valve is removed (Figure 3) (1).

Figure 3. Influence of the Vanquish SmartInject sample loop pre-compression technology on the recorded pressure trace during the run and more important on the retention time precision on a selected peak (benzophenone). Method: phenone separation in a gradient in 3.6 min from 40% to 100% acetonitrile and a Thermo Scientific Hypersil GOLD 1.9 µm 2.1 x 30 mm column. Flowrate: 0.26 mL/min, temperature: 25 °C, injection volume: 1 µL, detection: UV at 254 nm.

Why Is High Resolution Important for Mass Accuracy? High mass resolution is particularly important for all types of experiments involving complex mixtures, such as samples generated from a matrix (e.g. biological, environmental, food), since these contain a significant number of background (matrix) ions in addition to the possible analytes of interest. In such cases, high mass resolution makes the difference between detecting analyte molecules at low concentration and not detecting them due to the masking effect of isobaric matrix interference. Only with sufficient spectrometric resolution can a reliable and accurate result be achieved where analyte signals are separated well enough either from interferences or co-eluting compounds (Figure 4). 148 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 4. Two co-eluting compounds are only separated well enough at a resolution of 70,000 or higher, guaranteeing a reliable detection under all circumstances.

Figure 5. Thiabendazole measured and simulated data with resolution setting of 140,000 at m/z 200. 149 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.


The most striking feature of high mass resolution is the ability to directly determine the identity of the elemental composition of an ion signal based on the m/z value determined from the mass spectrum (Figure 5). Firstly, the accurate determination of the monoisotopic mass (A) peak restricts the pool of possible elemental composition combinations significantly. Secondly, high mass resolution in combination with accurate-mass measurements enables the user to directly depict fine structures, which further eliminates possible elemental compositions. For routine analysis, of course, in addition to hardware capabilities, appropriate software solutions are necessary to perform data acquisition, data processing and result reporting thereby providing an easy to use, streamlined workflow.

Figure 6. Schematic workflow diagram for the targeted and untargeted analysis of environmental samples.

Sample Processing Workflows When a given sample is analyzed together with a dilution series of a mixture of reference standards for the components of interest, the data can be processed in a targeted manner and quantitative results for the components of interest can be achieved. At the same time, non-targeted processing can be applied to the data obtained, with two different approaches for different purposes (Figure 6). The first approach is the suspect screening, where a list of components is taken as reference and used for data processing. Here, for all components given in the suspect list, extracted ion chromatograms are created which can be checked for the existence of a chromatographic peak. Usually, the suspect list 150 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.


contains additional information like retention time under given chromatographic conditions, the elemental composition, and fragment masses of the suspect compounds. So from the elemental composition the theoretical accurate mass can be calculated and used for a first identification of the compound. At the same time, a theoretical isotope pattern can be calculated and compared with the experimental one found in the chromatographic peak for confirmation of the first identification. If MS/MS spectra are available, the fragment information can be used for additional confirmation. Additionally, built in spectral libraries are another option for result confirmation. This then leads to the identification of certain compounds in the sample which forms the final screening result. If reference standards for the identified components can be obtained, the qualitative result can be transformed in a quantitative one. The second approach is the non-target screening, or General Unknown Screening (GUS). In this case, results are not obtained from a list of components, whatever size it may have, but they are obtained from the sample itself by applying an unbiased search algorithm that creates extracted ion chromatograms from every single mass in any spectrum acquired for a given sample and checks these for the existence of chromatographic peaks. This process is called feature detection. Grouping these features into components by sorting out isotope signals and possible common adducts yields the initial screening result of the non-target screening. Result confirmation can be obtained by generation of the elemental composition using the isotopic pattern of the found components and online searches in databases like ChemSpider or online libraries such as mzCloud. Prerequisite for these processing approaches is the ability of the LC system to separate as many components as possible chromatographically and to deliver retention times of highest reliability. The mass spectrometer then needs to separate the remaining co-eluting components (analytes and matrix components) into distinct spectral signals, since this is the only guarantee that the measured mass is the correct one for a given analyte of interest. Thermo Scientific software packages provide all three stages of data processing: targeted quantitation, targeted (or suspect) screening as well as non-target screening. It can all start from a full scan detected water sample, where known components, which have certified standards available, are able to be quantified. At the same time, other components can already be identified using the same identification criteria, namely retention time, fragment ions with ion ratio, and isotopic pattern, followed by fragment and library search on MS2 data. In addition, a non-target screening can be run in combination with targeted screening or as stand-alone processing, again providing multiple stages of confirmation. An unbiased detection algorithm (“feature detection”) mines the data and generates a list of compounds that are present in a sample and compares multiple samples with respect to the existence of these components in all samples. Mass spectrometric signals need to be grouped as isotope signals and common adducts to avoid multiple detection of the same component. Chromatographic alignment takes care of slight retention time differences that might occur due to differences in sample load and matrix composition. For data mining differential analysis and multivariate analysis are important tools to reduce the amount of data that is generated during feature detection. As shown in Figure 7, features 151



are represented well in the scores plot, which points to differences in samples, as well as in the loadings plot, that shows compounds that are responsible for the differences in the samples.

Figure 7. Example for scores and loadings plot in an environmental analysis.

Online searches are a very important part of compound identification in nontarget screening analyses, one of the most commonly used being ChemSpider™. Another offer here is the mzCloud online database, created and maintained by Thermo Fisher Scientific.

Network-Based Instrument Control and Data Processing In order to help develop harmonized strategies, it is useful to have a networked data system. This enables the instruments to be in one location, control is then available from elsewhere. In addition to this data processing, reporting and data storage can be performed from a separate location. Thermo Scientific™ Dionex™ Chromeleon Chromatography Data System (CDS) software is ideally suited for this purpose through its client/server architecture, and the administration of such a network is centrally controlled, thereby ensuring data security in addition to simplifying the administration tasks. An example of this could be seen at any water treatment facility attached to a manufacturing site. It is necessary for such a plant to monitor its effluent prior to discharging this into the environment. However it is likely that all necessary analytical equipment is in a central laboratory on site, in order to save costs and concentrate the expertise in one place. With a networked data system, the samples can then be taken to the central laboratory and placed in the sample queue following any necessary preparation steps. Once this is done, a user from anywhere on site can operate the instrument and perform the analysis prior to processing the data and providing a report including the RTI. The availability of the data in real time local to the treatment facility or anywhere else on site enables any action to be taken quickly should it be necessary. 152



Using eWorkflows™ To Enable the Fastest Route from Sample to Results The development of harmonized workflows has been discussed by a number of authors in previous sections. Once these workflows have been developed it is necessary to propagate them throughout the relevant community in order that the analysis is conducted in a similar manner in all laboratories. Chromeleon eWorkflows™ take advantage of the fact that all chromatography workflows are similar; injection, separation, data (signal) capture, result generation. It is only in the details of these steps by which any two workflows would differ from one another. The key benefits of creating eWorkflows are as follows: 1. 2. 3. 4.

The quality of data is consistently high The results are consistently reported The eWorkflows™ themselves can be easily distributed to other users Once distributed, using an eWorkflow™ requires no special training, thereby saving costs

In order to create an easy-to-use eWorkflow™, it is necessary to know any instrument methods, processing methods, and report formats. In addition to this, the basic structure of the sequence, any documents to attach (e.g. SOPs), and how the sequence name should be generated are required. The various methods and documents are entered initially to the new eWorkflow along with a description. Once this is done, the defaults for these methods are chosen, plus electronic signature settings and sequence name generation. The final and most important step is to define the sequence structure. eWorkflows split a sequence into four blocks; a header, sample block, bracket, and footer. The header is a set of samples always run at the beginning of the sequence; likewise the footer is a set run at the end. They will normally consist largely of standards in order to ensure stable operation of the instrument. The sample block and bracket are then used to determine how many samples can be run before it is necessary to run a standard, and how often this can be repeated in any given sequence. Once this has been entered, the eWorkflow can be saved and used.

Conclusion With modern instrumentation and software even non-target screening tasks become more and more applicable even for laboratories that are more orientated to routine work and do not have as much time and resources than pure research laboratories. Development of high-throughput methods without sacrificing robustness is a growing demand from environmental laboratories. These should satisfy the need for reducing analysis time in a field where the number of samples and compounds per samples are increasing every day. The method of today has to cope with the need of tomorrow. And the analysis of today must already contain the information that may be required tomorrow. In this regard, MS instruments capable of sensitive target quantifications and HRAM non-target 153 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.


full scan acquisitions at the same time will allow the data they produce to be re-processed in the future in order to look for new compounds of public and government interest, without repeating the analysis. Indeed, regardless of the time and resource consumption that repeating the analysis can bring, in some cases it may no longer be possible, as samples may not be available, or they may have degraded. At the same time, further developments aim to reduce the manual work for sample preparation and the time spent on data acquisition and processing with the use of authomated tools, as the eWorkflow, in order to decrease the source of errors and increase productivity. The improvement of easy and effective normalization strategies, as the RTI, will help to share data and knowledge among laboratories and institutions.

References 1.

2.

3.

Paul, C.; De Pra, M.; Martin, M.; Sneekes, E. J.; Bailey, B.; Steiner, F. Is Retention Time Precision Only Affected by the LC Pump? Poster Note PN71518-EN 0215S, Thermo Fisher Scientific, 2015. https:// tools.thermofisher.com/content/sfs/posters/PN-71518-LC-Retention-TimePrecision-Pumps-Pittcon2015-PN71518-EN.pdf?elqTrackId=6e3eaaede 7614b27b5100c68381eac53&elqaid=1619&elqat=2 (accessed June 2016) How Solvent Delivery Technology Can Improve Confidence in Peak Identification and Quantification SP71188-EN 0714S; Thermo Fisher Scientific, 2014. https://tools.thermofisher.com/content/sfs/ brochures/SP-71188-Solvent-Delivery-Highest-Confidence-PeakIdentification-Quantification-SP71188-EN.pdf?elqTrackId=97d9450580 d44d908c72085db85bcb95&elqaid=1619&elqat=2 (accessed June 2016) Grosse, S.; Greco, G.; Letzel, T. Retention-time-index (RTI) – a novel chromatographic parameter in the field of LC-MS(/MS) – An interlaboratory study; ANAKON 2013, Conference Proceeding. http:/ /www.bmbf.riskwa.de/_media/RISK-IDENT_Statusseminar_TUM.pdf (accessed June 2016)

154 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.

Target, Suspected-Target, and Non-Target LC-MS ... - ACS Publications

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