Dr. Michael Jarvis: Hi. Welcome to today's webinar by AB SCIEX. And I'm introducing here today the new AB SCIEX 4500 series LC/MS/MS systems and their applications in clinical research and forensics toxicology.

So, here we have the new AB SCIEX Triple Quadrupole 4500 and QTRAP 4500 LC/MS/MS systems, which are the instrument of choice for clinical research and forensic toxicology laboratories that require dependable, integrated workhorse LC/MS/MS solutions to deliver fast, sensitive results that they can trust hour after hour and day after day.

Now, this new system includes many of the features that make its big brother, the 5500 series mass spectrometer, so popular, and these include: an integrated valve, an integrated syringe pump, the legendary Turbo V ion source, and a very small footprint which is only 32 inches by 32 inches in width and length and 24 inches high. This system also supports up to 180 pounds of UHPLC equipment on top of the mass spectrometer. So, both this feature and the small footprint make this system ideal for laboratories where space constraints are a concern.

Now, the 4500 system has big shoes to fill. It builds on the legacy of the API 4000 system, which was arguably the most successful triple quad ever. This new system builds on the legacy of the API 4000 by setting a new benchmark for reliable quantitation with at least 10 times greater sensitivity over all other triple quadrupoles in the same class. The published specification for 200 femtograms reserpine on column is a signal to noise of 2,000 to one.

Now, there are many new innovations that result in improved performance in the 4500 series mass spectrometer. The first of these is the QJet 2 ion guide. Now, the QJET 2 ion guide is an RF-only quadrupole, which effectively focus ions into the Q0 region of the mass spectrometer. And what this does is it captures more of the ions than a traditional skimmer cone would capture. Allowing more ions and less gas into the Q0 results in much higher sensitivity.

In keeping with the tradition of the API 4000 system, the 4500 series of the mass spectrometers has an excellent robustness, and this is in large part due to the Turbo V ion source. This robust source features an orthogonal spray design and supports flow rates of up to five mils per minute. It also allows easy to use plug-and-play electrospray ionization and atmospheric pressure chemical ionization probes.

In the slide presented here, we can see robustness test for the compounds methamphetamine and amphetamine. Fifteen hundred injections of plasma were made over seven days, and we can see that the internal standard corrected peak areas of these compounds were less than 3 percent and less than 2 percent respectively for amphetamine and methamphetamine.

Another key feature of the new 4500 series LC/MS/MS systems is the new vacuum system with only one small turbo pump. In comparison, the 4000 series had two turbo pumps. The new interface design of the 4500 series with QJET 2 ion guide enables much more efficient pumping. This new vacuum system is not only more robust and reliable for more productivity, but is a much cooler and lighter system.

Another improvement in the new 4500 system is the EQ electronics. The upgraded electronics allow improvements in both speed and extended mass range. In terms of speed improvements, this system supports four times faster tandem quadrupole scan speeds compared to the API 4000 with no loss in quality in precursor ion and neutral loss scanning modes. .

This system also supports fast MRM measurements with dwell times of as low as one millisecond and pause times as low as one millisecond. Furthermore, this fast system supports positive naked ion polarity switching in 50 millisecond switching time.

In terms of mass range improvements, the new RF quill box [sp] design with a drive frequency of 940 kilohertz provides a mass range of up to 2,000 Daltons. The higher frequency better confines the ion beam and especially improves the sensitivity for lower mass ions.

And finally, there have been improvements made in the Linear Accelerator trap technology. These improvements include axial field within the linear ion trap which move ions towards the extraction region. Also improved is the ion extraction itself and much more efficient in-trap fragmentation.

The faster scan speeds and cycle times of this instrument allow scan rates of up to 20,000 Daltons per second with better than unit resolution. There are also much shorter in-trap fragmentation times; all of these allowing this instrument to run well in compatibility with UHPLC methods.

Another key feature of this instrument is the curved LINAC collision cell. This space-saving design allows for a smaller instrument footprint. The new Linear Accelerator design is what enables the short dwell and pause times and the fast scanning in MS/MS mode. Furthermore, the curve of the LINAC collision cell helps to eliminate neutrals, preventing noise on the detector.

This system also features new detector technology for quantitation. The accurate pulse-counting detection system counts pulses of ions impinging on the electron multiplier for a timed period, creating digital representation of ion flux. This system is less susceptible to noise than analog detectors. Furthermore, it does not require filtering and thresholding of data. All of this means that we get better detection of low level signals with the highest possible accuracy.

Now, let's look at a few examples of the performance of the new AB SCIEX Triple Quad 4500 system. In this first example, we'll investigate the quantitation of six compounds, along with six internal standards, in a single method using the MRM mode.

Comparing the sensitivity of the new 4500 system to the 4000 system, we can see that on a compound-dependent basis we get improvements in sensitivity that range from one to approximately three times in intensity. If we scale the data for the 4000 QTRAP system to the same axis, we can see that the improvements on the QTRAP 4500 and Triple Quad 4500 system are quite significant.

On this slide, we have a summary of a direct comparison of the sensitivity in MRM mode, comparing the Triple Quad 4500 system to the API 4000 system. As you can see the gain in signal for these five compounds range from 1.1 times to 2.4 times, with an average gain in signal of 1.6 times. All in all, the AB SCIEX Triple Quad 4500 system delivers equivalent or better sensitivity compared to the API 4000 system on a compound-dependent basis; nevertheless, we expect that, by and large, we will see performance gains.

And just to demonstrate the analytical and quantitative performance of this new system, this slide demonstrates the linearity, accuracy and CVs [sp] for quantitation in MRM mode using one of the example compounds that we were investigating in the previous slide. For the antiretroviral compound, m-tryptamine [sp], over the range from five to 5,000 nanograms per mil, we can see that the accuracy is never more or less than 10 percent deviant from the expected accuracy, and the CVs range from one up to 7 percent.

In this second example, we investigate the use of the QTRAP 4500 system for the purposes of drug screening. In this example, we've taken urine and spiked it with a mixture of drugs. We monitor 508 MRM transitions simultaneously, two transitions for each of the compounds under investigation. Because of the Turbo V ion source, we're able to run the system at a flow rate of 2.2 mils per minute. In this particular experiment, we will leverage the speed of the QTRAP system to perform fast, sensitive scanning, allowing collection of high quality MS/MS spectra for every compound detected.

In this slide, we can look at some of the data collected with the method described previously for the analysis of drugs in urine using the QTRAP 4500 system. This analysis was performed using a Phenomenex Kinetex C18 column and a simple dilute-and-shoot sample preparation. We can see that while screening for 508 transitions simultaneously we have detected the presence of a number of compounds here, and we have simultaneously generated EPI, or enhanced product ion, spectra for these compounds. We have representative spectra for hydromorphone, maparadine and fentanyl.

Not only does the QTRAP 4500 system give us incredible gains in speed, it also provides incredible gains in sensitivity when operating in linear ion trap mode. On the left hand side, we have comparable data acquired using a 4000 QTRAP system with the MRM survey scan on the top and the IDA-triggered MS/MS data on the bottom. When we compare to the same experiment performed on the QTRAP 4500 system, we can see that we have a moderate gain in sensitivity in MRM mode as expected, but we have an enormous gain in sensitivity in EPI, or enhanced product ion, mode. Much more signal, much cleaner spectra, which means much better library searching confirmation.

And in this third example, we investigate the possibility of performing positive-negative switching with scheduled MRM. In this example, urine has been spiked with a number of drugs, including pain panel drugs, barbiturates and the metabolite of THC. We know that the pain panel drugs will fly in positive ion mode, while the barbiturates and the THC metabolite will fly in negative mode. The speed of the 4500 system allows us to monitor both the positive and the negative compounds simultaneously using the scheduled MRM algorithm.

On this next slide, we can see an example of the quantitative performance of this system while running a positive-negative switching experiment performed using the scheduled MRM algorithm. We use buprenorphine as a representative compound, and we can see that the excellent linearity over a range of 0.25 to 1,000 nanograms per mil is more than acceptable. On the right hand, we've tabulated the percentage CVs and the accuracy over the entire range. We can see that the accuracies range from 84 percent to 117 percent over this range, and the percent CVs are no worse than 12 percent at the low end of the concentration range.

The 4500 series LC/MS/MS systems deliver more than just a tandem mass spectrometer. These are just one part of a total solution. The AB SCIEX accelerated lab integration package for clinical research combines the 4500 series with the Eksigent Expert UHPLC systems, the Cliquid software, iMethod applications, multiplexing capabilities with online solid-phase extractions, and training into a comprehensive workflow specific to high-throughput targeted quantitation.

So, in conclusion, the 4500 system delivers the world's most popular triple quadrupole intelligently reengineered. This fit-for-purpose design offers best-in-class performance with equivalent or better sensitivity in MRM mode compared to the API 4000 system. This system offers a mass range up to 2,000 Daltons. Many speed improvements have made this system compatible with the fastest chromatography, and these include: the fast dwell and pause times of one millisecond; the 50 millisecond polarity switching times; fast quadrupole scan speeds of 12,000 Daltons per second; even the faster QTRAP scan speeds up to 20,000 Daltons per second. The QTRAP has a 50 times improvement in sensitivity in QTRAP scanning mode compared to the existing 4000 QTRAP. Furthermore, the speed and sensitivity improvements on this platform lead to vastly improved quantitation in MRM queued mode.

And finally, we have several improvements, such as the integrated syringe pump and valve and smaller footprints, that lead to improved usability of this system.

Thank you very much for your attention.