Geoff Harper reports on some innovative – and unusual – applications of chromatography technologies
The versatility of chromatography is well known and every day new ways of achieving early diagnosis of a variety of conditions and diseases are being developed. Both in academia and industry, research has been ongoing to extend knowledge and capability in answer to medical need.
Life’s a gas
The use of chromatography for diagnostic purposes has been an industry standard now for a few decades. The main settings it is used in are clinical laboratories (such as pathology/biochemistry departments) all the way through to the groundbreaking research facilities more commonly linked to university research departments. Ian Parry, UK manager, gas chromatography(GC) & gas chromatography - mass spectrometry (GCMS) at Shimadzu UK explains that chromatography allows the user to separate out individual components and to identify them based on a number of criteria (LRI, retention time, mass spectral matching). “It is unique in its ability to find a match for a component by simply asking a commercially available or research library for a list of possible hits – all done via software control - whereas in times past, the user had to go to an actual paper library to search for their results.”
GC is for more volatile components such as organic acids and steroids, whereas liquid chromatography (LC) is more suited to use in volatile components such as vitamin D, says Parry. They are typically used to determine patient levels to identify if there is a condition already present, or if a biomarker can be found to show that a patient may be pre-disposed to a certain condition developing.
“Established techniques, such as liquid chromatography - mass spectrometry (LCMS) and GCMS are now being replaced with liquid chromatography-tandem mass spectrometry (LCMSMS) and gas chromatography-tandem mass spectrometry (GCMSMS) because the inclusion of the extra MS capacity allows the laboratory to see lower and lower levels, by using the power of the MS units in tandem, but also to increase the accuracy of the result given. MSMS techniques monitor transitions of compounds rather just individual ions, which can lead to the potential for false positives. An example of this would be the use of GCMSMS in the detection of drugs of abuse. The limits of detection can be lowered and components can be identified with greater accuracy by using MSMS versus MS alone.”
Parry says that Shimadzu products have grown with the market demand – the need to see lower limits with greater accuracy, and also to separate out greater complex mixtures of components in single analytical runs. The adaptability of Shimadzu products not only refers to the hardware, but also the reporting software used to allow users greater flexibility in how they can report their findings. Product development is always looking for new ways to make analytical testing an easier, less time consuming and more specific way of identifying compounds of interest. An example of this is MALDI-TOF – an imaging technique that allows identification and structural characterisation of biomolecules.
“The ability to identify components now by MS has been a massive leap forward, and the ability now to identify accurate mass to 2dp and above is a huge leap from where we were – let’s hope the next years see radical changes of similar magnitudes.”
HPLC
High-performance liquid chromatography (HPLC) has proven by far the most versatile form of chromatography separation in the clinical laboratory, explains Lisa Thomas from Thermo Fisher Scientific. The technology emerged in clinical chemistry labs in the 1970s and is ideally suited for separating complex biological samples such as blood, urine, and plasma. Today the majority of lab-developed tests applications are for the analysis of amino acids, peptides, proteins, carbohydrates, lipids, nucleic acids, vitamins, and hormones.
Traditionally the most common detection technique coupled to an HPLC was ultraviolet (UV) or fluorescence technology. However, since 2005, there has been an exponential increase in clinical research publications referencing HPLC triple quad mass spectrometry (LC-MS/MS). MS-based methods for various lab developed tests have become increasingly popular because the technology offers the flexibility to develop cutting edge in-house tests to measure clinically relevant compounds, as well as metabolites, and small volumes of samples with confidence. The coupling of HPLC and MS, can offer in the space of a few minutes separation, identification and quantification of hundreds of analytes in a complex biological matrix.
Clinical laboratories are gradually increasing the volume of lab-developed tests traditionally done on HPLC with UV detection or GCMS technology because it’s generally faster and cheaper in the long run.
“In the future, leaders in the clinical laboratories will continue to see more analysis moving to HPLC MS- based workflows moving toward larger molecule analysis of peptides and proteins,” says Thomas. “Most promising clinical research areas continue to be in endocrinology, inborn errors of metabolism, and therapeutic drug monitoring research. With the focus on precision medicine, medical scientists continue to ensure they can better understand how to administer the right treatment at the right time for the right person in the right amount; particularly for disease areas such as cancer.”
For complex biological samples, Thermo Scientific uses its TurboFlow, which Thomas says improves MS detector response to analytes of interest (for instance, testosterone) by removal of non-analyte matrix interferences such as phospholipids, which can interfere with the measurement of the analyte subsequently improving signal to noise ratio. These methods often save several work-flow hours by eliminating several manual steps
This automated technology offers rapid exclusion of large molecules such as proteins as the smaller analyte molecules stick to the nearest surfaces inside the pores of the particles used to pack the TurboFlow columns. Other sample components that don't stick very much, such as salts and sugars get rinsed away quite rapidly. The entire loading step of a typical method takes only 30 seconds.
It takes time to wash and equilibrate columns between injections. Multi-channelling is a way to reclaim that time so that the detector is not waiting too long for the next relevant data acquisition event to come by. It basically involves scheduling the autosampler to inject into the available channels of the system in a staggered fashion so that the detector can collect data from each at the appropriate time while the other channels are getting cleaned and equilibrated. Multi-channelling across two channels doubles sample throughput as long as the data window time is less than half of the total run time.
The Prelude MD HPLC instrument uses syringe pumps that Thomas claims offers several advantages over conventional HPLC pumps. The system contains a dedicated pump for each solvent bottle. There are very few moving parts to wear out. There is no routine pump maintenance required by the user. If necessary, a service engineer easily and quickly replaces a pump by swapping modules.
Academic research
Meanwhile, in the UK, scientists at the University of Liverpool and the University of the West of England at Bristol have developed a system, Odoreader, which responds to signature chemicals emitted from urine, much in the same way as dogs detecting certain cancers in humans through their keen sense of smell.
The device consists of: a chamber for heating the sample (urine) that releases the smell; a chromatography column, which spreads out the compounds responsible for the smell; a sensor, an electric device that has a variable resistance that changes as gas compounds pass over it, and an algorithm that interprets the change in resistance of the sensor to detect patterns linked to disease.
Professor Chris Probert reveals that the inspiration for the device was two-fold, “Firstly our clinical observation that the faeces smell differently in the presence of disease (so we started to look at that) – secondly, the observations that dogs can respond to the smell of skin cancer (melanoma) and bladder cancer.”
Probert was based in Bristol for 20 years and set up the work with Professor Norman Ratcliffe, then he moved the lab to Liverpool. He reveals that they have recently been awarded funds to conduct a much larger study of bladder cancer to determine whether the results are reproducible. So far the team has achieved 95% accuracy with new data due in two to three years
The team has a five-year plan that will culminate in a product launch for bladder and prostate cancer. The intention is an accurate, affordable device for diagnosing urological cancer and a range of gastrointestinal disease, he says
New diagnosis in New Delhi
Chromatography has also recently been used by a team at the All India Institute of Medical Sciences, New Delhi, in the identification and purification of the overexpression of MSMB and PSA, used in the diagnosis of prostate cancer.
“Chromatography is basically a technique to separate and purify the molecule of interest,” explains assistant Professor Subhash Chandra Yadav, co-author of the paper Overexpression and purification of folded domain of prostate cancer related proteins MSMB and PSA, which outlines the research. “Many chromatography techniques were well in practice depending upon the molecule of interest such as ion exchange, hydrophobic interaction. The chromatography techniques used in this paper to purify the PSA and MSMB from recombinantly overexpressed was affinity chromatography. This is entirely based on the ligand binding capability of genetically introduced His Tag in these proteins, which bind to Ni metal on Ni-NTA chromatography material. Combining with the spectroscopy, these proteins can be identify (western blotting, etc) and quantify (absorbance, Bradford reaction, etc).”
Much of the technology for the identification and quantification were already globally in practice but there is always a scope for improvement. A common example: ELISA was well-established technology for the quantification of ligands in human pathology (Extensively used for PSA detection) but is limited as regards sensitivity and specificity. Thus innovations were introduced to overcome these problems.
“Innovations are gradually making these technologies more efficient and cost effective,” says Yadav. Most of the research is starting to produce cost-effective, simple and effective (high sensitivity and specificity) diagnostic technology.
