|SEND TO PRINTER|
Feature Articles : Feb 15, 2009 ( )
Boosting Potential LC/MS Operations
“Pittcon 2009” Will Highlight Emerging Trends in Both Industry and Academia
Although mass spectrometry (MS) dates back to 1897, and high-performance liquid chromatography (LC) to the 1970s, scientists are still developing methods to enhance their capabilities. Rapid advancements in the pharmaceutical and biotechnology fields are the main drivers behind new developments. Many efforts are stemming from academic labs, which are opening new avenues for these technologies.
LC/MS is now a multibillion dollar business with tandem LC/MS comprising the majority sector. In this article, a handful of researchers provide a preview of some of the topics that will be covered at “Pittcon 2009” next month.
A researcher in the University of Michigan’s chemistry department is developing analytical methods to study changes in metabolites within pancreatic islets of Langerhans cells. Charles Evans, Ph.D., says that this involves developing new liquid-chromatography methods, miniaturizing LC separations using capillary columns and multidimensional separation.
“By developing higher-resolution separation methods, we can more effectively take a complicated sample and break it up into all its components,” he says.
Islet cells contain beta cells, which are responsible for secreting insulin and maintaining constant blood glucose levels. “We are studying the metabolites present in these beta cells, which may be involved in pathways associated with diabetes development when they fail and are unable to secrete adequate insulin quantities.”
The multidimensional, 2-D separation involves two different liquid chromatography columns. A standard separation is performed first on one column and the fractions collected. These fractions are further analyzed in the second column; the columns must be different.
“Select two different columns that have as little correlation as possible between the separation on the first dimension as on the second dimension,” Dr. Evans suggests. For example, perform reverse-phase separation as one dimension and ion-exchange separation as the second dimension.
He says that the metabolites they are primarily interested in detecting are those in central carbon metabolism, glycolysis, and the TCA cycle. Additional energy transfer metabolites like NAD, NADH, ATP, and ADP can also be measured.
“One change we detect is that there are more central carbon metabolites present in the cells as a response to stimulation with glucose. We also detect changes in the energy metabolites. We’re still sorting out what all these changes mean,” states Dr. Evans.
The overall goal of this research is to develop methods to help improve the understanding of the biochemical mechanisms that underlie Type 2 diabetes. “We’re hoping that being able to measure these compounds will detect changes associated with diabetes along the course of the disease.” Dr. Evans adds that this method could have potential use for many other components—whether in disease research or understanding biochemical mechanisms that change based on environmental factors.
Ionic liquids have an advantage over organic solvents like methanol in that they don’t denature proteins as much and enable protein separation in their native confirmation. This is the driving force behind the research of Neil Danielson, Ph.D., professor of chemistry at Miami University, Ohio.
“We have been separating small molecules like caffeine and aromatic carboxylic acids by reversed-phase liquid chromatography (RPLC) using ionic liquid modifiers like ethylammonium formate (EAF) and methylammonium formate (MAF) as replacements for methanol in the mobile phase.”
Furthermore, Dr. Danielson has discovered that MAF has an advantage over EAF in that it has a lower viscosity, which makes it more compatible in terms of pressure limitations. “Organic solvents have a very low viscosity, below one centipoise (cP). MAF has a viscosity around 9 or 10 cP, which is really good for ionic liquids. This gives us better efficiency in LC if we work with a less viscous mobile phase.”
Polarity of the mobile and stationary phases, as well as that of a sample, are all involved in controlling retention of a sample compound and the separation of a mixture of sample compounds.
“We can control the polarity of the mobile phase—the amount of MAF in water. This ratio will vary the polarity, which is effective in controlling the retention of many pharmaceuticals. If we increase MAF, this lowers the retention.”
In addition, his group discovered that using low levels of MAF (1%) to replace methanol, for certain compounds like warfarin, the methylammonium ion formed an adduct and enabled easier qualitative identification by RPLC with MS detection.
When using MAF at higher concentrations (5–20%), Dr. Danielson says his group is able to control retention of water-soluble vitamins and antibacterials. “This is quite novel in that we are the first to show this ionic liquid as an organic solvent replacement in LC/MS is compatible because it’s forming volatile components in the interface.”
He adds that a compelling use of MAF is as a major mobile-phase component of LC/MS that has been shown to provide an advantage for separation of proteins as well as antibacterials. However, he says, “we’re still searching for a clear advantage for the use of MAF for small molecules.”
Better Analysis of Biofluids
In response to the current trend to develop more potent drugs with lower circulating levels, there is an ongoing effort to develop methods to increase detection sensitivity in biofluids. “Our research is focused on sensitivity in bioanalysis,” says Paul Rainville, applications chemist with Waters. One way to increase sensitivity is with smaller particles.
Waters developed the use of sub-2-micron chromatographic particles in 2004, which can increase the speed of analysis and reach up to a 10-fold increase in sensitivity. The particles consist of a patented material, bridged ethyl hybrid (BEH) that can withstand extreme pressures and operate in a wide pH range.
The advantages of using an elevated pH mobile phase include selectivity gains previously unattainable; ability to chromatograph basic analytes in a neutral state leading to improved peak shape; and the promotion of ionization of molecules that are to be detected by mass spectrometry—enabling further increases in sensitivity.
“This doesn’t eliminate steps, but it does increase the speed of running your assay without reducing chromatographic performance. Our particles maintain separation quality and are done faster, and this means increased productivity and less expense,” states Rainville.
One of the current hurdles with LC in bioanalysis is to provide a solid, inlet platform for the mass spectrometer. It must have compatible flow rates and the ability to resolve matrix interferences from the drug or metabolite undergoing quantification. “This is why we developed our sub-2-micron particles and the hardware to go around it,” he adds.
Better sensitivity also enables correct determination of the fate or pharmacokinetics of a drug molecule—both important parameters for the success of a drug. The ability of instruments to perform tasks that address regulatory concerns such as sample-matrix monitoring or metabolites is also advantageous. Rainville says that this method will soon be applied to detecting steroids in biofluids, and may lead to developing new packing material to address this. “We’ll apply the sub-2 micron particles and elevated pH for any drug that may have a challenging limit of detection.”
Phosphorylated Proteins in CSF
In-depth analysis of cerebrospinal fluid (CSF) has the potential to reveal important details and malfunctions of many nervous system diseases. Dean Stuart, from the University of Cincinnati’s department of chemistry, and colleagues, are using different instruments to obtain better information from CSF components. Samples obtained from the university’s medical school were divided into three groups to compare various phosphorylated proteins and/or peptides: patients with post-subarachnoid hemmorhage, patients with arterial vasospasms, and normal patients.
“The overall goal would be to see if there’s a difference in phosphorylated proteins across that batch, and if so whether you could say that high levels of these proteins are a precursor to stroke or vasal spasm. I can see slight differences in phosphorylation using inductively coupled plasma MS (ICPMS) as the phosphorus specific detection and then using ion-trap MS to get structural data, followed by database-search software to figure out the identity of phospholated proteins,” explains Stuart.
In addition, using size exclusion chromatography, he was able to show a slight difference in phosphorylated types. This was done using a 5 kilodalton filter, to exclude anything larger than 5 kd. “We wanted to deal with small proteins or peptides, and get rid of big macromolecules like albumin.” Then, instead of fractionating the samples first, he used a size exclusion column with a range of 100 to 7,000 daltons for molecular weight exclusion. The resulting peaks were run on ion-trap MS and run through software (Agilent’s Spectrum Mill) for identification.
This size-exclusion method mimics conventional proteomic approaches. “In my case, the two dimensions are size exclusion chromatography followed by nano-reverse phase. I would hope this could be used further to find other phospholated proteins or any other type of proteins. The methodology ought to work for anything,” Stuart summarizes.
Identifying Drug Metabolites
A lab within the department of chemistry at Purdue University is developing methods for metabolite identification. “We first started developing these as a compliment to established methods of identifying functional groups in an unknown analyte,” explains Steven Habicht, a scientist working in the lab. He says there are certain situations where identifying specific impurities is not possible by MS/MS alone, and the more additional compounds, the longer it takes for analysis and the more expensive it is.
This HPLC-MS/MS method is based on ion-molecule reactions for identifying tertiary N-oxide functional groups in unknown analytes. “This is applied early in the drug discovery phase, but it could also be used for stability-indicating assays to see if any compounds will be formed when exposed to high heat, humidity, or from storage in plastic containers. If extra products are formed from the compound, you need to identify what those are and whether they are toxic.”
In order to introduce neutral molecules into a commercial quadrupole ion trap MS, his group developed an external reagent mixing manifold. This allows tri (dimethyl amino) borane (TDMAB) to be mixed with the helium buffer gas used in the trap. The analyte of interest is isolated in the trap and reacts with TDMAB for a specific time. The reaction delivers only protonated N-oxide analytes as they elute from the HPLC column. This process was demonstrated using Clozapine N-oxide and Olanzapine-N oxide.
“We’re creating our own niche in metabolite identification with these methods,” states Habicht. “There are situations where you can’t distinguish two different types of metabolites where these ion-molecule reactions can fill the void.” He adds that the main focus is to continue to develop a library of different reagents to make functional group identification a straight-forward process.
There is little doubt that many more potential applications for LC/MS will continue to be developed as more targeted drugs become available through the efforts of genomics, proteomics, and metabolomics. LC/MS may become standard for clinicians searching to identify sources of side effects or potential toxins. Who knows where these technologies will lead medicine over the next 100 years?
© 2012 Genetic Engineering & Biotechnology News, All Rights Reserved