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.