Mr. Adrian Taylor: Hello and welcome to today's Webinar by AB SCIEX, where I'd like to introduce you to the new AB Sciex TripleTOF 4600 LC-MS/MS System and its applications in forensics toxicology

The AB Sciex TripleTOF 4600 System makes routine high-resolution accurate mass MS and MS/MS available to any laboratory

It's uniquely integrated comprehensive qualitative exploration, rapid profiling, and high-resolution quantitative workflows all available in one single workhorse platform

In this Webinar today, I would like to demonstrate some applications of the TripleTOF 4600 System. And this will include target screening and identification as well as high-resolution accurate mass quantitation and non-target screening, which incorporates general unknown comparative screening workflows

Before I do that, however, I'd like to introduce some of the performance attributes of this system, which have come about from the innovations of this instrumentation

The first one is the high sensitivity in both MS and MS/MS modes. This gives us MRM-like sensitivity as well as the ability to detect very low analytes of interest

A second attribute is high resolution, where we can have higher than 25,000 resolution in both TOF MS and TOF MS/MS scan modes

So, this gives us advantages, obviously, when it comes to qualitative information. But, it also gives us advantage when it comes to quantification

This allows us to select very narrow extraction ion chromatograms. And so, this gives us advantages when it comes to selectivity, especially for compounds in very complex matrices

Another attribute is its fast acquisition rates. So, here, we're able to perform fast acquisitions in both MS and MS/MS modes. For example, we can do up to 50 MS/MSs in a second, using our information-dependant acquisition mode

So, with this fast acquisition rate, this gives us the advantage that we can have more MS and MS/MS data overall. And this gives us high-quality information and increases in identifications

It also means that we have compatibility with fast chromatography. And overall, with these fast acquisition rates, we don't have any compromise in resolution

So, another attribute is high mass accuracy. And this gives us the advantage, where we can have definitive identifications

Another attribute is that we can get up to four times--four orders of linear dynamic range

And when it comes to adapting this technology of accurate mass quantitation and qualitative workflows in the laboratory, we need to concentrate on providing in intuitive software solutions. And this is what we've worked hard to provide with this instrumentation

So, one of the key goals in the development of this instrument was to achieve the speed and sensitivity of a triple quadrupole so that the instrument would have very strong quantitative performance

So, what we've done here with the TripleTOF 4600 System is that it's based on the AB SCIEX TripleQuad 4500 System

So, here, the ions from the source first transits through the larger-diameter orifice of the instrumentation and then go into the Qjet region

The Qjet provides increased sensitivity and robustness through improved ion containment. So, this quadrupole area operates at high pressure and provides better collision focusing and improved ion transmission

The ions are further caught and focused in the Q0 region of the instrument and then passed into the first mass-analyzing quadrupole

Here, after this, we have a collision cell, which is high pressure line at collision cell. And this accelerates ions through the collision cell and provides fast transit times for fast MS/MS and multiplexed analysis

Also, the advantage that we have here is true collision-induced fragmentation, which provides reliable and information-rich spectra

So, the accelerator TOF analyzer was designed to deliver the speed and sensitivity required for triple quad-like quantitation. But, in this instance, we also have high resolution and mass accuracy for qualitative applications

So, if I'm continuing down the ion rail, the ion compression optics behind the collision cell direct the beam into the accelerator, which is operating at 30 kilohertz

And so, by operating at this speed, we improve both the TG cycle and instrument dynamic range. So, the accelerator voltage of the TOF region is very high, at 10 kilovolts. And this provides high sensitivity, especially for the higher-mass ions

We also have a two-stage reflectron mirror. And this is used to compensate for any energy dispersion and also maximizes our resolution

And finally, we have a 40-gigahertz multi-channel TBC [sp] detector. And this provides very high sampling speeds. And we can maintain a high resolution even at low mass

So, when all of these technologies are combined, we really do have a best of both worlds in this instrumentation. So, here, we have the AB SCIEX TripleTOF 4600 System with qualitative analysis power of a high-resolution accurate mass analyzer as well as a quantitative speed, sensitivity, and linear dynamic range of a triple quadrupole instrument

So, this slide shows the high resolution and excellent mass accuracy that we can obtain across the mass range for selected drug compounds. So, the new AB SCIEX TripleTOF 4600 System provides high resolution of up to 35,000 dependent on the mass detected and also gives the stable mass accuracy of lower than 2 PPM at fast acquisition speeds in both MS and MS/MS mode

So, with the advantages that we have with high resolution and accurate mass, this gives us the ability to perform very narrow extracted ion chromatograms. So, this gives us both selectivity gains as well increased signal to noise

And we can see this from this slide here when going from a 1,000 PPM to 10 PPM mass accuracy. We're actually getting an 8 times gain in signal to noise

So, here, we detailed the experimental conditions that were used to demonstrate the applications of the TripleTOF 5600 in forensic toxicology workflows. So, we used high-flow chromatography with a kinetics column. And the mass spectrometer was operated in electrospray ionization and was performed automatic mass calibration

The TOF MS was operated with a 10-millisecond accumulation time. And we performed information-dependent acquisition where we acquired up to 20 dependent TOF MS/MSs using a collision energy of 35 volts with a collision energy spread of 15 volts

So, PeakView software with the XIC manager add-in was used for targeting and non-targeting data processing

And so, here, the XIC manager manages large lists of compounds and performs extracted ion chromatogram calculations, both targeted and non-targeted peak-finding operations as well as library searching

And so, the XIC manager allows the ability to review results based upon retention times, accurate mass, isotopic pattern, and MS/MS library searching

So, the PeakView software can further be used to aid in our compound identifications by giving us the ability to form both formula and structure elucidations as well as elemental composition determination and fragmentation interpretation so that we can competently identify unknowns

And then we have our MultiQuant software, which gives us the ability to quantitate a high number of analytes, both using full-scan MS and MS/MS data types

So, now, I'm going to show you some of the applications when it comes to targeted screening using the TripleTOF 4600 instrument

So, here, all we have to do is, after opening the data file in PeakView software, the XIC manager can be launched from the XIC manager menu in the PeakView toolbar

And here, the table contains a number of columns, which values can be edited, including the name, formula, the adduct or modification, the retention time and width

And to define an extracted ion chromatogram, a mass has to be entered. And this can done directly, or we can actually use the software to calculate the mass based on formula isotope or adduct provided or by pasting values from a spreadsheet

And the generated XIC list can be saved to future processing

So, here, we have an example of some data being processed by the XIC manager. And the results are displayed to show the mass error, the retention time of the analytes where it was found, and library searching results

So, any XICs above a defined intensity threshold are highlighted in green. And so, this figure demonstrates where we have 259 drug compounds that were extracted and identified from a spiked equine urine sample using information of the isotopic pattern of the detected molecular ion as well as retention time and accurate mass of the MS/MS spectral searching

So, this was where we've been able to search against a forensic drug MS/MS library for added competence in our identification

So, this confidence data for each compound identified can be visualized using traffic lights. And these confidence data can be actually user definable, as we can see from this slide here

So, further to what I've already previously explained, we can use IDA-triggered accurate mass MS/MS spectrum of a drug. And this can be overlaid with a matched library spectrum as visual indication of the closeness of the match

So, here, we have the bottom right panel of this figure that shows an example of a spectral overlay comparison of a library versus an equine urine sample of terfenadine

The sample spectrum is represented as a blue trace, and the library is the gray trace. So, this is a nice visualization of the match or the closeness of the match that we're gaining when it comes to MS/MS library searching

So, all the drugs that have been identified in the previous slide are quantifiable using the MultiQuant software. And so, we can get excellent accuracy and reproducibility, as we can see from this slide here

And another application that we can perform using the TripleTOF 4600 is non-targeted screening and compound identification

So, the next slides will describe how this is achieved. And one example of this is by using a general unknown comparative screening workflow in which we can compare a sample with a control. And so, this allows us to narrow down to the--only the relevant compounds in the sample by comparing the TOF MS and MS/MS data of a sample with the data generated for a control

In this particular example, we're comparing an equine urine sample with an equine urine control. And all ions with 10 times higher intensity in the sample then in the control is reported

So, for further identification purposes, we can use other tools of the PeakView software. And here, we have the formula finder that uses high-resolution accurate mass information of the molecular ion as well as the adducts and isotopic pattern and fragment ion information to empirically calculate potential molecular formula. And this can be only achieved using both MS and MS/MS data

So, here, this slide actually shows that the calculated formula can be automatically search against ChemSpider and so that we can actually find potential matching structures

And here, a proposed structure that was--been obtained from ChemSpider can be confirmed by using the fragmentation prediction tool and PeakView software

And in this particular example, each of the eight peaks matched with 100 percent explainable MS/MS fragment ions for the fentanyl structure

So, in summary, I've been able to introduce the new AB SCIEX TripleTOF 4600 System with its high sensitivity as well as high resolution and accurate mass MS and MS/MS workflows

The system has very intuitive software solutions for both qualitative and quantitative data processing. The PeakView software with the XIC manager can screen for and identify compounds with confidence and complex forensic samples

And we can perform using the XIC manager a sample control comparison workflow that aids in narrowing down to the significant compounds in our unknowns

MultiQuant software can then determine the concentrations of the identified substances. And further processing using the PeakView software using formula inder and automatic online database searching as well as fragmentation prediction tools can in the end give us a very high confidence in the identification of our unknowns

And so, in this presentation, I've been able to give you examples of the targeted and non-targeted screening for drugs in forensic samples

And that concludes this Webinar. And I thank you very much for listening