Gas or liquid chromatography combined with mass spectrometry is widely recognized as a versatile and powerful technique for the investigation of complex chemical mixtures. A state-of-the-art accounting of progress in this area took place recently in Hong Kong at the conference on “New Horizons in Clinical Chemistry.” The meeting, organized by C. S. Ho, M.D., of the Prince of Wales Hospital, Hong Kong, had a strong focus on clinical applications and explored new advances in liquid chromatography technology coupled to mass spectrometry.
“Mass spectrometers as clinical tools are being increasingly adopted for a host of clinical applications by the global clinical laboratory community,” said Michael Morris, Ph.D., head of clinical business operations for Waters. According to Dr. Morris, current clinical assays harness only a fractional percentage of the power of the instrumentation so there is a large effort in place toward the development of new MS–based biomarker assays.
From the mid ’90s, with the analysis of acylcarntinites and amino acids from neonatal blood spots, high-throughput applications based on mass spec have been widely adopted. Since that time, this technology has played an increasingly important role in clinical analysis, to the point where it is common to find it in laboratories with applications for endocrinology, toxicology, and therapeutic drug monitoring.
“Now, new realms are being explored, and while they are still in the discovery phase, they promise to yield exciting new applications in the next decade,” Dr. Morris explained.
The rise of automation, combined with ease of clinical operations and the growing use of these systems in a nonspecialist environment have become motivating forces for companies engineering new, more user-friendly instruments. The concept of analyzing hundreds of samples per day on a single system was met with incredulity a mere decade ago, whereas, that is a minimum expectation today. As the needs of clinical laboratories continue to evolve, companies are looking to mass spec for new applications.
Classically, 95% of LC/MS technology has been applied to characterizing compounds with a molecular weight of less than 1,300 daltons. However, recent developments in biomarker discovery using more advanced types of mass spectrometers are generating data pertaining to multiplexed indicators of disease.
“With the emergence of protein biomarker analysis by mass spec, some significant sample preparation may be required,” Dr. Morris said. “This is due to the fact that the target compounds need to be broken down into their constituent peptides for accurate and precise quantification, and interfering contaminants need to be eliminated from the sample to ensure analytical integrity.”
Alan Wu, Ph.D., is chief of the clinical chemistry and toxicology laboratories at San Francisco General Hospital. At the meeting, he discussed the transitioning of his toxicology lab from gas to LC/MS, as well as the role of immunoassays and time-of-flight (TOF) mass spec in compound identification.
Most toxicology testing performed in hospitals is immunoassay based, an approach which is less than ideal for a number of reasons. There are many more drugs than there are immunoassays, and many compounds have similar structures. Highly specific antibodies can be raised to individual drugs but this increases the number of assays by an order of magnitude, and companies have not stepped up to improve testing accuracy.
According to Dr. Wu, immunoassays won’t become obsolete as they cannot be replaced. Immunoassays are very fast, 20 minutes at most, and they require no sample preparation. Incremental costs are quite low, under one dollar per assay. On the other hand, LC/MS requires at least an hour depending on whether there is on-line extraction, but this contrasts with GC/MS, which requires three to four hours.
Various mass spec tools provide a range of options for compound identification. Because LC/MS is based on the use of a polar mobile phase, many preliminary processing steps can be eliminated. But the advantages must be weighed against the higher instrument costs and the caveat that GC/MS provides more resolution than liquid chromatography. Another option is the addition of tandem mass spec to the platform because the mass spectrum can differentiate between co-eluting compounds utilizing parent-daughter transition ions.
According to Dr. Wu, TOF is the next emerging technology; it offers interesting advantages over both GC and LC. These latter technologies rely on fragmentation patterns, retention times, and library searches for identification, so they require preexisting knowledge. TOF can identify complete unknowns since it gives exact molecular weight to parts per million accuracy.
“You have to know where these things come off,” Dr. Wu continued. “We’re not interested in introducing fragments. TOF changes the paradigm because it gives precise molecular weight to 3–4 decimal points. You can determine the exact molecular formula of a target compound so that compound can now be found without knowledge of its retention time.”