Mr. Andre Schreiber: Welcome, everybody, to a Webinar of AB SCIEX about new solutions for food safety testing using the new TripleTOF 4600 LC-MS/MS System and intuitive data processing tools

Before I start today, I would like to introduce myself. My name is Andre Schreiber. I'm working for AB SCIEX in the Technical Marketing Group, and I'm responsible for method and application development with focus on food and environmental analysis

In today's presentation, I would like to introduce the new AB SCIEX TripleTOF 4600 System to you. I will talk about high-resolution accurate mass capabilities of the instrument, sensitivity, linear dynamic range for triple quadupole-like quantitation, and about MS/MS capabilities for compound identification

As part of my presentation, I would like to talk about software tools, especially our new version of PeakView, including the XIC manager, the formula finder, and the capability of automatically search the Internet for unknown chemical structures

I also will talk about MultiQuant, and I will highlight a couple of key applications of that instrument, specifically targeted screening and identification, high resolution and accurate mass quantitation, the capabilities of sample and control comparison, and some key software features and applications for general unknown screening

In this slide, you can see an image of the TripleTOF 4600 System and a list of typical key performance criteria. The instrument has very high sensitivity in MS and MS/MS mode, offers high resolution of more than 25,000 resolution in MS and MS/MS

It is very fast. It can acquire up to 50 MS/MS spectra per one second in IDA mode. Mass accuracy, we typically see less than two PPM in complex samples. Linear dynamic range for quantitation is typically up to four order of magnitude, and the instrument comes with lots of intuitive software solutions to easily adapt to accurate marks quantity and qualitative workflows

At the beginning, I would like to show you a couple of hardware components of the instrument. When we developed that mass spectrometer, we started with our triple quadrupole ion path of the 4500 System, which contains the Qjet ion guide, the high pressure Q0, the first quadrupole, and our linear accelerator condition cells for very fast MS/MS experiments

And then we added our accelerator TOF analyzer out of our 5600 System the top of the mass spectrometer, which contains ion compression optics. It is accelerating ions with 30 kilohertz and 10 kilovolt into the time-of-flight tube

At the bottom, you can see our two-stage mirror and that ions are reflected back to the top to reach our 40 gigahertz time to digital converter

And by combining the capabilities of the 4500 with the mass analyzer of the 5600, we create a very powerful quadrupole-quadrupole time-of-flight instrument, which combines qualitative results using high resolution and accurate mass with quantitative data based on speed, sensitivity, and linear dynamic range, which are more typically common for triple quadrupole analyzers.

In this slide, you can see an example of pesticide screening using an MS information-dependent acquisition MS/MS experiment. Here, we used full-scan time-of-flight MS to screen for pesticides being present in a sample

And the software automatically acquired MS/MS data at each point of the chromatogram. And this MS/MS data can be used for compound identification

At the bottom of the screen, you can see a couple of examples of resolution. And in the middle, there is the red box showing the mass error for these peaks across the mass range typical for pesticide analysis, starting from 200 all the way to more than 700

And you can see resolution values at these different masses. And the concentration and the standard we injected here was a 10 nanogram per milliliter pesticide standard using our ID quant kit

Based on high resolution and accurate mass information, we can now increase the sensitivity of detection of targeted analytes by extracting very narrow ranges of targeted ions from our full-scan chromatogram

And of course, at the moment when we make the narrow of the extracted ion chromatogram smaller, we automatically increase selectivity. And this is also decreasing our signal to noise

The example here on the screen is showing the detection of imidacloprid in a kale extract after 10X dilution

And you can easily see, at the moment, then we narrow the--at the range of the extracted ion chromatogram, we remove noise. We remove interferences. And this automatically is improving our limits of detection

Before I go into different data and applications, I would like to share some experimental conditions with you. We extracted our food samples using a QuEChERS procedure. And we diluted extracts by a factor of 10 with water to reduce possible matrix effects

We performed a UHPLC using a Shimadzu USLC System with a polar modified column from Restek and very standard gradient conditions based on water, methanol, and ammonium formate

We used the TripleTOF 4600 System with electrospray ionization. And we automatically calibrated the mass axis of the instrument using the calibrant delivery system between injections

And our method contains of TOF MS survey covering the whole mass range of interest. And then we automatically acquire TOF MS/MS data throughout the chromatogram with standardized collision energy. And we use these MS/MS spectra for compound identification based on mass spectra library searching

And data processing, we use the PeakView software together with the XIC manager and the MultiQuant software for quantitative data analysis

First, I would like to focus on targeted screening and quantitation. Before I start showing you data, I would like to introduce the latest version of the XIC manager to you

The XIC manager was designed specifically for screening, identification, and applications like typical food residue analysis. The XIC manager contains a table that you can see on the screen. And you as a user need to input the name of compounds, formulas, and retention types. And the software then is automatically calculating all the needed mass information to process the data

And as a second input, you have to input some confidence settings into the software with respect to mass error], retention time error, isotopic patterns, and MS/MS library searching

You can set these criteria. And depending on the results of data processing, this will then color traffic lights with whether green, yellow, or red to display and visualize your confidence scores

And here's an example of typical pesticide screening results in a food sample. This is an extract of a Clementine QuEChERS extract 10 times diluted. And I processed the data using the XIC manager. And the software is automatically reporting that it found thiabendazole and imazalil

It's reporting mass error, retention time error, and the library searching score. And all these are visualized in the green traffic lights you can see on the left side

And for further review, we can hit the review button on the software. And this will display the time of flight and S-signal and on the left side, our acquired spectrum overlaid with the theoretical spectrum. And it also reviews the MS/MS data. That's what you can see on the right side. That is the acquired MS/MS spectrum overlaid with our library spectrum

Here's another example of using the XIC manager to process data off a kale extract. In the kale extract, we found imidacloprid that was the pesticide I was talking earlier about

And also the software reported that dodemorph might be in the sample with excellent mass accuracy, a good retention time hit, matching isotopic pattern. But, if you look at the lower right side, the MS/MS pattern is not matching our library spectrum at all

This is resulting in a purity score of 17.9 percent only. And this clearly indicates that dodemorph is not present in the sample but some isobaric interference

And this is a clear example that MS/MS and fragment ion information is very crucial for compound identification. Without doing MS/MS analysis, there is a big chance to report false positive data

Another functionality of the XIC manager is the possibility to do sample comparison. And here, I used that feature for a quantitative comparison of two different samples

I compared an orange extract with a pesticide standard I injected. And then this case then the XIC manager is reporting the pesticides which are present in the sample

It was reporting thiabendazole and imazalil based on MS and MS/MS information. But, also, it tells me that these two pesticides are above the concentration I'm interested in by comparing to my pesticide standard

If I further want to quantify the amount of pesticides in my sample, I can quickly export the accurate mass data into the MultiQuant software, use injections at different levels to generate calibration curves, and can flow this way, calculate unknown concentrations in my samples

And here on the screen, you see an example of the calibration line of thiabendazole, one of the pesticides I found in the orange sample. You can see repeat injections at a concentration of 0.1 nanogram per milliliter on the top

At the bottom, you see the calibration line and also, in that little table in the middle, information about accuracy and reproducibility, which have been excellent over the linear dynamic range

With this, I would like to move over to non-targeted screening or for unknown screening and compound identification

Let me go back to our kale sample where the software gave us a good hit for dodemorph based on matching mass accuracy, matching retention time, and matching isotopic pattern, but where the MS/MS spectrum clearly told us that dodemorph is not present in our sample

So, I wanted to know actually what is present in the sample instead looking like dodemorph. So, I took the MS and the MS/MS spectrum, and I exported these two spectra into our formula-finding software that is also integrated in the PeakView software

Here, you can see a screen shot of the formula-finding software. The formula finder takes the information about mass accuracy of our molecular ion, the isotopic pattern, fragment ion information from the MS/MS spectrum, and empirically calculates molecular formulas based on that. And it's also looking into adduct formations to tell us what kind of molecular ion do we have

In this case, the software detected that we have a protonated ion. But, it could also report an M plus ammonium or M plus sodium adduct, for instance

Our empirical formula calculation was resulting in a single possible formula only, C18H35 and O

In a second then--in an automatic step, this formula is sent towards the Internet. And we perform a structure search in ChemSpider in the Internet. And the formula finder is returning a total of six possible structures matching that formula the formula finder was calculating

And you can see these formulas being listed at the bottom. And one of those structures is shown in the right side after I was clicking on that ChemSpider hit

After my ChemSpider search, I took these formulas which have been reported and imported them into the fragment prediction tool, which is another tool which is integrated into the PeakView software

And on the screen, I show two different structures which have been returned by the ChemSpider search. One is dodemorph, the pesticide the XIC Manager was finding before in my kale sample

And if I automatically compare the structure of dodemorph with the MS/MS spectrum, and that is shown on the top in the MS/MS spectrum, the software is not able to explain all the fragment ions in that MS/MS spectrum. You can see many of these fragment ions are color coded in red. That means the software is not able to explain these fragment ions based on the structure of dodemorph

If I do the exact same comparison of the structure of oleamide, a natural occurring fatty acid amide, with the MS/MS spectrum I recorded, the software can explain 100 percent of the fragment ions. So, that gives me another indication that actually oleamide was present in my kale sample but not dodemorph, as already suggested before by the library searching results

With this, I would like to summarize today's Webinar. I was talking about the high sensitivity, high resolution, and accurate mass capabilities of the new AB SCIEX TripleTOF 4600 System

This instrument comes with many different software features for efficient and easy qualitative and quantitative data processing. This includes the PeakView software, which contains the XIC manager, a very powerful and easy-to-use tool to screen for targeted and non-targeted chemicals in complex food samples

The XIC manager is identifying compounds with high confidence based on retention time, accurate mass, isotopic pattern, and MS/MS library searching

The XIC manager can also perform sample control comparison to give us quantitative information in specific samples. And we can export this data towards MultiQuant to perform quantitation using calibration lines of these identified compounds

Also, the PeakView software contains a number of features which are very powerful for unknown identification. And I was specifically talking about the formula-finding software, the automatic online database searching, and the MS/MS fragmentation prediction tool, which compares structures with the MS/MS pattern. And all these tools together allow us to identify unknown components and samples

And I was showing you a couple of examples of food samples from fruits and vegetables where I used all these powerful tools for targeted and non-targeted screening applications

With this, I would like to finalize today's presentation

I would like to say thank you for listening. Have a good day. Bye, bye