This article reviews recent developments that aim to address the conflicting demands for higher throughput, sensitivity, and selectivity in bioanalysis applications. 

Bioanalysis is a key part of a candidate drug's pharmacokinetic and pharmacodynamic characterization. Liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) has been used widely by pharmaceutical laboratories over the past 30 years to quantify drugs and their metabolites in biological matrices. Numerous advances in instrumentation, technology, methods, and software during this period have led to significant improvements in speed, and sensitivity alone has increased by seven orders of magnitude.

Analyzing drugs in biological samples has always been challenging as a result of matrix effects or background interference, and researchers have tried to overcome this in the past with various method developments and sample preparation steps. More recently, addressing the issue has been complicated further by the need to increase sample analysis throughput, while at the same time being able to detect compounds at increasingly lower levels. 

One approach has been to use ion mobility mass spectrometry (IMS), which combines ion separation techniques with mass spectrometry, including quadrupole or time-of-flight (TOF). IMS has been commonly used for the selective detection of compounds in biological samples.

  Ion mobility can operate at atmospheric pressure, unlike mass spectrometry, and this allows the incorporation of atmospheric pressure ionization.

  This enables additional selectivity during sample introduction, but IMS is typically too slow and not sufficiently rugged for quantitative bioanalysis applications.

The development of multiple reaction monitoring (MRM) with triple quadrupole mass spectrometers has gained broad popularity over the past 30 years for its significant advantages, including improved selectivity and quantitative accuracy. In MRM mode, two stages of mass filtering are employed on a triple quadrupole mass spectrometer. In the first stage, an ion of interest (the precursor) is preselected in Q1 and induced to fragment by collisional excitation with a neutral gas in a pressurized collision cell (Q2). In the second stage, instead of obtaining full scan ms/ms where all the possible fragment ions derived from the precursor are mass analyzed in Q3, only a small number of specific fragment ions (product ions) are mass analyzed in Q3. This targeted MS analysis using MRM enhances the specificity and, with that, the lower detection limit by up to 100-fold (as compared to full scan ms/ms analysis) by allowing rapid and continuous monitoring of the specific ions of interest. This approach became the gold standard for many pharmaceutical laboratories, and the more recent development in MRM

 achieved further improvements in selectivity, by increasing the number of fragmentation steps.

However, achieving the required levels of selectivity and speed for accurate bioanalysis remains challenging for even the most sensitive instruments. Typically, today's assays need to be able to detect compounds ranging from low ng/mL levels to pg/mL; at these levels it can be very difficult to separate out compounds of interest accurately from other compounds. Matrix interference can result in either unresolved peaks, or in high baselines. Unresolved components can be resolved through slower chromatography (longer gradients) or through additional sample clean-up, which is labour-intensive and slow, adding unwanted time and costs to the analysis. Pharmaceutical laboratories typically aim to complete each sample analysis in under two minutes, which is not possible with these extra steps, especially if peak integration then needs to be adjusted manually on a sample-by-sample basis. High baseline problems can have adverse impacts on the limits of quantitation (LOQ) and dynamic range.

Therefore successful drug quantitation in complex matrices requires much greater selectivity than can be achieved with traditional instruments and methods. With the growing interest in large molecule biotherapeutics and tightening regulations on drug characterization, new approaches for more selective quantitative assays are required. 

Recent developments in ion mobility technology have led to the development of effective differential ion mobility separation techniques, where the ion separation takes place in a cell integrated in the ion source region, directly in front of the curtain plate. Rather than separating ions in time as they cross the cell, the ions are separated in trajectory based on the difference in their mobility between the high and low field portions of the applied RF (see Figures 1 and 2).

  A second, compensation voltage is applied to correct the trajectory of the desired ions along the axis of the cell, while other ions will migrate away because of the difference in mobility.  

Figure 1: Analysis of clenbuterol in urine. (a) This assay suffers from matrix interferences in all three MRM channels. (b) Using effective differential ion mobility separation techniques, with isopropanol as a chemical modifier, resulted in the reduction or elimination of interferences in all three MRM channels.			

This results in a very robust, stable, and high-speed technique that enables the use of short MRM cycle times.

  It effectively provides orthoganol separation in between the LC and MS stages to remove background components, allowing accurate lower level compound detection.

  The separation can be further enhanced by the addition of chemical modifiers, such as isopropanol, to the gas phase.

Figure 2: Pentoxifyline in protein precipitated plasma. Very high baseline is successfully removed with DMS, resulting in a 20-fold improvement in signal-to-noise ratio at 10 ng/mL.			

Even with this approach, detection of compounds at the pg/mL level can still be challenging as ion suppression becomes a much greater issue. Another approach that is gaining interest for high throughput bioanalysis is combining micro LC with ion mobility MS. By using columns with diameters of under 1 mm, micro LC can offer fast separations with improved sensitivity. Micro LC also uses smaller diameter electrodes, which can minimize post-column dispersion, resulting in improved chromatographic resolution with narrower and higher peaks (see Figure 3).

Figure 3: Low flow generates true and reliable information. Adapted and reproduced with permission from Springer and the Journal of The American Society for Mass Spectrometry, 14(5), 2003, Effect of different solution flow rates on analyte ion signals in nanoESI MS, or: when does ESI turn into nanoESI?, Andrea Schmidt, figure number 1. Â© Springer.			

For bioanalysis applications, the lower flow rate and smaller diameter columns of micro LC help to increase the sample efficiency (or the ionization efficiency), which reduces ion suppression considerably. It also means that less sample needs to be injected, which also enhances sample efficiency and lowers ion suppression. Using micro LC also means that at least 10 times less solvent goes into the mass spectrometer (see Table 1 in reference 7), reducing solvent consumption, as well as reducing the costs, space, and paperwork associated with transporting hazardous waste products.

Advances in MRM technology offer scope for improvements in resolution and performance, but instrumentation companies will continue to strive for better selectivity, enhanced sensitivity, and higher throughput. Further challenges will also arise from trying to keeping pace with newer bioanalysis methods, such as emerging trends in micro-sampling and micro-dosing (cassette dosing). These techniques have the potential to significantly reduce the sample volumes required in bioanalysis. For example, capillary blood microsampling can use just 8–10 μL blood for a single time point,

  but implementing these techniques introduces a number of new technical challenges.  

2. A.B. Kanu, P. Dwivedi, M. Tam, L. Matz, and H.H Hill Jr, 

4. Y. LeBlanc, D. Caraiman, M. Aiello, and H. Ghobarah, AB SCIEX technical note, publication no. 2960211-01 (2011). 

5. J.A. Ferguson, P. Clemens, and L.Y. Olson LY (2013). "Evaluation of differential mobility techniques for quantitative and qualitative identification workflows in pharmaceutical environments," Poster #265 at the ASMS 2013. 

Journal of The American Society for Mass Spectrometry

Microsampling in biopharmaceutical analysis

 (Future Science Ltd., London, UK, 2013). 

9. Cen Xie, Dafang Zhong, Kate Yu, and Xiaoyan Chen, 

10. L. Dillen, W. Cools, L. Vereyken, W. Lorreyne, T. Huybrechts, R. de Vries, H. Ghobarah, and F. Cuyckens, 

11. K. Mriziq, S. Hobbs, T. Settineri, and D. Neyer, Eksigent application note, publication no. 4590211-01 (2011). 

has worked in the pharma space for 30 years, both within large pharma companies, and with instrument suppliers. He is an analytical chemist by training and has a PhD in advanced characterization of complex mixtures. This gives Steve considerable practical as well as theoretical knowledge of chromatographic and MS systems and their integration. The first 25 years of his career were spent characterizing small molecules, and in the last five years he has followed the industry trend and moved on to the characterization of biopharmaceuticals. For the last two and a half years Steve has been the Market Development Manager for the pharmaceutical business for EMEA with AB Sciex. 

http://www.absciex.com/applications/drug-discovery-and-development-mass-spec

 This article is from The Column. The full issue can be found here:

http://images2.advanstar.com/PixelMags/lctc/digitaledition/August22-2014-uk.html

Articles

Features

This article reviews recent developments that aim to address the conflicting demands for higher throughput, sensitivity, and selectivity in bioanalysis applications.

Recent Developments in Ion Mobility Mass Spectrometry for Bioanalysis

Incognito attends a sushi class and ponders the plight of the analytical scientist.

Recently while on summer vacation I found myself taking a class on sushi making — something I'd wanted to do for several years. The class was excellent and the chef/instructor was a very amiable young man who was keen on talking about the "chemistry" of the sushi-making process — the "pickling and fermentation" of the rice, the release of starch as you patted the rice down on the Nori paper that helped to bind the rolls etc.  

Photo Credit: Skip ODonnell/Getty Images			

As we were working away, I couldn't help but notice that among some very ornate and brightly coloured tattoos which the chef sported on his forearms, there was a tattoo of what I thought I recognized as styrene. My first thought was that my new chef acquaintance had been misled and that an internet trawl for interesting sushi-based chemicals had gone badly wrong, and he would be forever doomed to be tagged with a popular monomer rather than something much more pertinent, romantic, or even illegal...one never knows! 

My curiosity eventually got the better of me and in a quiet moment at the end of the class I asked the chef the relevance of his tattoo. He went on to reveal further molecules on his arms, which I've drawn out in the following figure.  

There's a prize for anyone who can guess the relevance of these molecules at this stage, without reading further ahead. 

Well, from left to right in the figure the molecules are acrylonitrile, 1,3-butadiene, and styrene. When polymerized they form the very common thermoplastic terpolymer, acrylonitrile butadiene styrene, more popularly known as ABS. So chef, it turns out, knew more than just a smattering of chemistry and thankfully was not labouring under a misapprehension about his indelible markings.  

But why ABS? Well, it turns out that brightly coloured tattoo inks are made, at least in part, from ABS, which makes the colours extremely vivid and certainly explained the very brightly coloured fish and other tattoos that the chef was also sporting. He had though it pertinent to pay homage to the chemistry behind his skin art. 

But let's get to the point of this instalment. 

It turns out that the chef was a highly enthusiastic analytical chemist, who had spent some considerable time working in laboratories during his undergraduate programme and had initially taken a job as a trainee sushi chef to help fund himself through college. Indeed, he had graduated, undertaken a Master's degree, and had begun to work in a laboratory (a subsidiary of a multi-national corporation) that I had visited several years ago, and which was undertaking, at that time, some good research using reasonably advanced techniques. That's where the good news ended. 

It turns out that my chef friend and many of his classmates were highly disillusioned with analytical science and while he obviously had a love and passion for the subject, he earned about 30% more as a sushi chef. His benefits package was also much better working for the sushi chain. 

Furthermore, he went on to describe how his cohort, with whom he spoke regularly, all felt like second- or third-class citizens behind the chemical engineers, organic chemists, and biochemists with whom they worked. There was a common attitude that the analytical guys were there to simply turn the handle and generate data, which the "higher ups" would then interpret and contextualize to produce "information" useful to the business.  

The final straw for chef was the quality of the food in the subsidized staff restaurant! His free sushi was too much of a draw and he left analytical science after around a year in the laboratory. 

Ok — this was a young guy (and some of his friends) with only a very small amount of relevant experience, but does any of this resonate with you? Have you ever felt like a second-class citizen, employed to churn out mountains of data, which a superior being will then turn into something useful for your business? 

I have to say that at times, and in certain jobs within chemical analysis, I have felt like that. It is at these very times that I have taken it upon myself not to turn to sculpting rice and fish into a tasty dinner, but to learn absolutely everything I could about the way in which my data is produced and its relevance to my internal or external customers; to make sure that the results are fit-for-purpose and will result in the insight required to take the project forward, or not, as the case may be. In short, to care enough to go the extra mile. Now this takes time, often personal time, and a good deal of what employers these days call "discretionary effort" and I can in no way guarantee that it will improve your benefits package to something more than a sushi chef might expect. However, if you really do have a love for analytical science, in your next "sushi moment" instead of looking for a way out, how about looking for a way further in? It might just make life in the lab a little more appealing than life at the fish slab. 

My Sushi Moment

 Journal of Agricultural and Food Chemistry 

highlights the potential use of rosemary and oregano in the management of Type 2 diabetes mellitus (T2DM) (1). T2DM is typically managed using a combination of diet control and drugs to inhibit specific targets associated with T2DM; however, herbs contain high levels of bioactive compounds that could have the same therapeutic effect.

The ability of four herbs — Greek oregano (

) — to inhibit dipeptidyl peptidase IV (DPP-IV), a drug target of T2DM, was investigated. Mexican oregano and rosemary was shown by liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI–MS) analysis to contain the phytochemicals eriodictyol, naringenin, hispidulin, cirsimaritin, and carnosol. In biochemical inhibition assays, cirsimaritin, hispidulin, and naringenin were the most effective DPP-IV inhibitors. 

According to the authors this work demonstrates the potential use of herbs in the management of T2DM but further work is needed. — B.D.


  1.         A.M. Bower, L.M.R. Hernandez, M.A. Berhow, and E.G. de Mejiya, 

Journal of Agricultural and Food Chemistry

News

A new study published in the Journal of Agricultural and Food Chemistry highlights the potential use of rosemary and oregano in the management of Type 2 diabetes mellitus (T2DM).

Type 2 Diabetes Bioactive Compounds in Rosemary and Mexican Oregano

When a scientist needs to search through scientific literature, which is the preferred search engine? While there is quite a bit of variety in available search engines, 

 Blogger Kevin Schug has a definite preference. Here's what it is, and why. 

I am not the most computer savvy individual, but through a lot of practice I have become pretty good at searching the scientific literature. If you ask scientists, it does not take long before you realize that there is quite a bit of variety in the different search engines that they prefer. These days you can basically search anything or any content on any site. For finding papers on a subject, biologically oriented people tend to favour PubMed, but I have never really been a fan because it always seems difficult to find the way to directly get to the full paper. I think a lot of people like to search directly on Science Direct. Given the number of journals indexed there, that approach seems to work fairly well. In fact, in the past I liked to use a search engine called Scirus, which was run by Elsevier and would easily allow you to select whether you wanted it to search for PubMed or Science Direct articles. I would always filter by Science Direct, because more often than not it provided an easy path to full articles from journals for which my institution had a subscription. Search results could be easily refined as you went along. I recommended Scirus quite a bit to my students and colleagues; however, late last year I went to visit Scirus and encountered a notice that said it was going to retire. I believe that it actually mentioned that a reason for this end had something to do with the success of Google Scholar, but I am not sure if I just imagined that or not. In any case, I was compelled to go and check out Google Scholar, and I have to say that there are many things that I really like about it — it is definitely my new "go to" search engine for scientific literature.  

I like Google Scholar not just for its ability to search, but for its ability to collate my papers through a personalized page, to collate papers into a library on a particular topic I set, to track citations of my articles, and especially to send me email alerts when new articles are published that contain keywords that I have specified. Although I have not fully refined my alerts, one that I set on "hydraulic fracturing or unconventional drilling" has been extremely beneficial for keeping me up to date on that quickly evolving field of research. Of course, I am most interested in aspects of this topic related to environmental analysis and impact, but I have also been alerted to really interesting articles peripheral to this focus. I have enjoyed reading about the economic benefits of unconventional drilling. I have enjoyed studying about how the rest of the world is carefully evaluating how it can responsibly engage in this practice. I have enjoyed learning more about how the engineering has evolved to continually improve unconventional drilling. I believe this might be the first time my group has entered on the ground floor of a rapidly evolving field of research;

  it is absolutely crucial that I am constantly apprised of new publications that can inform and develop our work. This alert system certainly does the job — even this blog article will now appear in a forthcoming email alert to me. 

I receive three other email alerts, and these also have some unanticipated benefits. One on "antibacterial drug discovery" brings a lot of interesting information on both the chemical and biological side of the topic. I am working together with others to develop an innovative educational programme where freshman students at U.T. Arlington are collectively engaged in research in their first semester. The topic for the initial cohort, which I will help direct, will be natural product drug discovery, so this alert keeps us aware of the very newest research in the area. Communicating the absolute newest breakthroughs to the students should be extremely motivating and engaging for them. Another alert is based on "transmission mode–desorption electrospray ionization mass spectrometry" (TM-DESI), which is an ambient ionization technique we have helped develop recently.

  Initially, I set this alert because very little work has been performed using this technique (the approach was first reported by the Brodbelt group at U.T. Austin

), and I wanted to know if any other groups started to use it. As far as I know, no one else has done much work using TM-DESI, but I have also been pleased to find out that this search string consistently yields a number of interesting articles in mass spectrometry research that I did not anticipate showing up in my alerts. For example, this keyword combination brings up a lot of the recent work that is published on matrix-assisted laser desorption–ionization (MALDI) mass spectrometry. It also provides some interesting coverage of other ambient ionization techniques that I might otherwise miss. So, even though I have not received any alerts that pertain specifically to TM-DESI (of course, I would if they were published), I still have been reluctant to change or alter the search string because of the ancillary information it has provided. 

The last search string is one that I have altered — and I did so rather quickly after I received the first set of alerts. I set an alert for "restricted access media", because we have been doing a lot of work using this material to facilitate on-line sample preparation of biological fluids.

  Restricted access media combine size exclusion properties and standard chromatographic retention properties on the inner pores of particles. Now, interestingly, despite the exceptional performance of these materials, their use for on-line sample preparation still has relatively limited use compared to other sample preparation strategies, so I receive few alerts directly on this topic. That said, my initial setting of "restricted access media" brought some very unanticipated information. My first alert contained a cross between research on access to pornographic websites and freedom of the press; there was absolutely no chemistry, much less analytical chemistry, contained in any of the articles listed. While interesting, this result was a little further away from what I was looking for, and I frankly was a bit worried if some topics were found by others to be present in my work email. To remedy this situation, I quickly altered the alert request to "restricted access media and chromatography". That seemed to fix the situation, but I do still receive some fairly uninteresting articles (to me) that are listed simply because they are posted in open access journals and somewhere mention the word chromatography. I will be excited as some recent submissions from our group become published in the

. I am co-editing a special virtual issue on sample preparation for mass spectrometry in 

				 together with Prof. Michael Laemmerhofer, and two of the articles that will be published in the issue feature our recent work on restricted access media. 

So, for sure, I simply have to keep refining my search approach. At first I felt dismay for the demise of a search engine that I had grown to be familiar with and trust. Yet, as I changed to new technology, I have to say that I have been nothing but pleased. I am sure that there are other excellent search engines out there, and even though I have not heard of them at this point, perhaps one day I will be compelled to use them. But for now, I will stick with Google Scholar; if you have never used it, I recommend you check it out — and if you receive some highly unanticipated search results from your keyword searches, let me know about them. I am always up for a good laugh. Cheers! 

1. B.E. Fontenot, L.R. Hunt, Z.L. Hildenbrand, D.D. Carlton Jr., H. Oka, J.L. Walton, D. Hopkins, A. Osorio, B. Bjorndal, Q. Hu, and K.A. Schug, 

2. S.H. Yang, E.H. Wang, J.A. Gurak, S. Bhawal, R. Deshmukh, A.B. Wijeratne, B.L. Edwards, F.W. Foss Jr., R.B. Timmons, and K.A. Schug, 

4. S.H. Yang, H. Fan, R.J. Classon, and K.A. Schug, 

http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1615-9314/homepage/sample_preparation_in_mass_spectrometry.htm

is an Associate Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington, USA. He joined the faculty at UT Arlington in 2005 after completing a Ph.D. in Chemistry at Virginia Tech under the direction of Prof. Harold M. McNair and a post-doctoral fellowship at the University of Vienna under Prof. Wolfgang Lindner. Research in the Schug group spans fundamental and applied areas of separation science and mass spectrometry. Schug was named the 

 Emerging Leader in Chromatography in 2009, and most recently has been named the 2012 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science awardee. 

Cover Story

When a scientist needs to search through scientific literature, which is the preferred search engine? While there is quite a bit of variety in available search engines, LCGC Blogger Kevin Schug has a definite preference. Here's what it is, and why.

The LCGC Blog: Unanticipated Benefits of Keyword Searching the Scientific Literature

Issue PDF

Click the title above to open The Column August 22, 2014 North American issue, Volume 10, Number 15, in an interactive PDF format.


Vol 10 No 15 The Column August 22, 2014 North American PDF

Click the title above to open The Column September 22, 2014 Europe & Asia issue, Volume 10, Number 15, in an interactive PDF format.


The Column-08-22-2014

Recent Developments in Ion Mobility Mass Spectrometry for Bioanalysis

My Sushi Moment

Type 2 Diabetes Bioactive Compounds in Rosemary and Mexican Oregano

The LCGC Blog: Unanticipated Benefits of Keyword Searching the Scientific Literature

Vol 10 No 15 The Column August 22, 2014 North American PDF

Vol 10 No 15 The Column September 22, 2014 Europe and Asia PDF