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Food testing and forensic laboratories want to maximize productivity, deliver better-quality data, and remove the element of risk associated with manual interpretation. The right software is therefore integral to analysis.
SCIEX OS software for the X-Series QTOF accurate mass, mass spectrometers (SCIEX, Warrington, U.K.) boosts turnaround time and maintains high-throughput levels. It has been engineered to process complex data and present it in a user-friendly, navigable format, while alleviating potential pitfalls attributed to manual interpretation.
As drugs and pesticides continuously undergo synthesis, laboratories require a more reliable analysis method. This article depicts the SWATH Acquisition (SCIEX) workflow as an alternative to assays performed on triple quadrupoles as applied to food testing and forensic analysis laboratories. Until recently, SWATH Acquisition had not been widely used for the detection of pesticides in food samples or complex matrices in urine and blood samples.
Figure 1 shows the SCIEX OS control hub. The tiled layout creates a familiar navigation format for the operator. From this control hub, under the acquisition section, users can build MS methods and LC methods, create batches, and run samples. Data processing is performed using the explorer and analytics functions for simultaneous identification and quantitation. Instrument management can also be controlled for instrument suitability checks and to adjust user settings and privileges.
Figure 1 – The SCIEX OS interface allows users to acquire, analyze, and report in one place.SWATH Acquisition
SWATH Acquisition is a data-independent acquisition (DIA) strategy. When combined with the sensitivity and high-speed MS/MS acquisition enabled by SCIEX QTOF hardware, virtually every detectable analyte in the sample is fragmented and captured for true MS/MSALL. As the name suggests, users get a complete MS and MS/MS picture of all analytes that are detectable in the sample, without the need for costly repeated runs and time-consuming reanalysis. SWATH Acquisition using SCIEX OS provides a formidable tool for forensic drug screening and food safety.
In forensic applications, SWATH Acquisition eliminates the risks of missing crucial analytes that may not be detected on conventional targeted analytical workflows. SCIEX OS software captures a full complete digital archive of the sample, resulting in the following measurable cost savings.
Digital archive
Sample storage costs can be expensive and, depending on the type of testing a lab performs, the sample may need to be kept for several years. Despite advances in refrigeration and sample preservation techniques, there is still a risk of sample degradation. Using the SWATH workflow in SCIEX OS allows users to take a sample snapshot at that moment in time when the sample is in its truest form to initial condition just after sampling. The impact of creating a digital archive has numerous benefits across all forms of LC-MS/MS analysis.
Forensics
SCIEX OS was designed exclusively for the X-Series QTOF systems so that operators can acquire data, analyze results, and publish reports, all within the same interface for enhanced productivity. SWATH acquisition was employed to perform a forensic drug screen to look for 85 common illegal drugs and novel psychoactive substances.1 For this study, blank urine samples were spiked with multiple drugs commonly found in forensic settings at different concentration levels. With a significantly low run time of fewer than 3 minutes using SWATH Acquisition, researchers generated a true profile of the sample. The rapid workflow delivers a comprehensive drug screen, producing data that can be used for quantification, qualification, and screening. Figure 2, taken from SCIEX OS, shows the short gradient applied to this study. Mobile phase A was 10 mM ammonium formate in water, and mobile phase B was 0.1% formic acid in methanol. Separation was achieved using a Synergi Hydro-RP column (20 × 2 mm, 2.5 μm) (Phenomenex, Torrance, CA).
Figure 2 – Gradient conditions in SCIEX OS for chromatographic separation.The MS/MSALL facilitates all the confirmation parameters required, such as MS/MS spectra, ion ratios, and library confirmation. Also in this study, to add contrast, the SWATH workflow was performed against a longer gradient of 6.5 minutes. The results between the rapid gradient and the longer version are comparable. Table 1 shows their performance across 17 unknown spiked samples.
Table 1 – Performance of the 6.5-min and 2.5-min SWATH workflow for accuracy in detection
Food safety
The multiple reaction monitoring (MRM) approach to food safety has long been established as a daily technique used globally by food testing laboratories. Using MRMs, laboratories can quantitate how much of an analyte residue, such as a pesticide, is present in a sample. Although MRM analysis is powerful, an element of uncertainty remains. To ease the risk of reporting data, which may be subjected to doubt, SWATH Acquisition using SCIEX OS on the X500R is being adopted by food laboratories analyzing infant food, for example.2 Using this acquisition mode in SCIEX OS, food safety labs can scan for every detectable residue in a sample rather than just the compounds on a targeted list.
Simultaneous quantitation and qualification can be achieved using high-resolution accurate mass.3 A typical sample preparation protocol for fruits and vegetables requires no change for SWATH Acquisition for food analysis. The QuEChERS method was used to extract samples of leek, cauliflower, bean, jujube (after washing), jujube (not cleaned), and pear. The chromatographic element of the experiment remained the same, as did the mobile phases, column, and gradient profile one would expect to use on an LC-MS/MS system.
The desired mass windows and energies were set in the experiment field to build the SWATH Acquisition MS method. Figure 3 shows the criteria selected to perform a comprehensive TOF MS/MS scan.
Figure 3 – Criteria, including mass range and applied energies, used in the creation of the MS method.Once the sample is acquired, the data can be reviewed and interpreted. Unlike conventional MRM approaches, SWATH Acquisition has acquired all detectable analytes in the sample. When processing the data, the critical information needed from a sample is clearly displayed.
Key indicators can be quickly reviewed (see Figure 4). The traffic light scoring system, for example, enables the user to instantly see whether the mass error, retention time, isotope, and library confirmation meet the specified criteria to determine a positive or negative result. Other data shown in the analytics include the actual peak of interest, which will be automatically integrated but can be manually adjusted depending on the analyst’s interpretation. Acquired spectra are displayed and contrasted against the expected spectra from the vast pesticide library. Ion ratios are also reported for further confirmation. Therefore, this approach supplies all the information users need from a triple-quadrupole instrument workflow, yet with additional information for significantly greater confidence in reporting of results.
Figure 4 – Analytics interface of SCIEX OS for data review and processing.The acquired data quantified the detected pesticides using a 190-compound standard mix from which a set of calibration standards was produced and analyzed. Table 2 shows the pesticides and concentration of the residue (ng/ mL) found in the six vegetable and fruit extracts.
Table 2– Pesticides detected and their associated concentration (ng/mL) in the various extracts
Conclusion
As can be seen from the two examples using the SWATH Acquisition workflow within SCIEX OS, it is possible to transition MRM workflows from LC-MS/MS triple quad systems to high resolution accurate mass instruments such as the X500R QTOF. The sensitivity and robustness of the instrument enable it to handle the real high-throughput challenging matrices demanded by forensic and food safety laboratories.
References
- https://sciex.com/x69118.xml
- https://sciex.com/x50738
- https://sciex.com/x69198.xml
Phil Taylor is with SCIEX, Phoenix House, Lakeside Drive Centre Park, Warrington WA1 1RW, U.K.; tel.: +44 1925 236060; e-mail: [email protected]; www.sciex.com