CHROMATOGRAPHY 33
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.
“Te ability to identify components 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 we see radical changes of similar magnitudes in the coming years.”
HPLC High-performance liquid chromatography (HPLC) has proven by far the most versatile form of chromatography separation in the clinical laboratory, explains Lisa Tomas from Termo Fisher Scientific. Te technology emerged in clinical chemistry labs in the 1970s and is ideally suited for separating complex biological samples such as blood, urine
UK-based scientists have developed the Odoreader, which responds to chemicals emitted from urine
and plasma. Today the majority of lab-developed test applications are for the analysis of amino acids, peptides, proteins, carbohydrates, lipids, nucleic acids, vitamins, and hormones. Traditionally the most common detection technique
C New diagnosis in New Delhi
hromatography 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 work 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 identified (western blotting, etc) and quantified (absorbance, Bradford reaction).” Much of the technology for the identification and quantification was 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
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
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.
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