by Pauline Griffeuille and Dr Sylvain Dulaurent
Mass spectrometry (MS) can be combined with a number of chromatographic separation and ionization methods, which results in powerful technologies for both qualitative and quantitative molecular analysis. CLI caught up with Dr Bhattacharyya (Thermo Fisher’s Senior Manager for Clinical Research and Toxicology) to find out more about the past, present and potential future uses of MS in clinical science and diagnostics.
Can you provide a brief introduction to mass spectrometry, please?
Mass spectrometry (MS) dates to over a century ago to 1918, when J.J. Thompson ionized neon and separated the ions using magnetic and electric fields. For many years after, MS was used to detect isotopes and simple elements. The advent of novel ionization techniques such as electrospray and desorption-based methods enabled researchers to develop the ability to detect biomolecules, which quickly escalated to the development of analytical methods for disease detection and therapeutic treatments.
Clinical laboratories have been using immunoassays for a long time for the identification and quantitation of analytes in biological matrices. However, immunoassays rely on the availability of highly specific antibodies and can have cross reactivity challenges, which can result in poor selectivity and reproducibility. With clinical laboratories also seeing a significant increase in the number of analytes and the complexities that every scientist needs to address, immunoassays cannot always meet the requirements of an analysis or workflow. From small to large molecules, from analysing one analyte to hundreds in biological matrices (ranging from drugs of abuse to therapeutic drugs), the analytical results are expected to be accurate, sensitive, robust and reliable. Liquid chromatography (LC) coupled to MS methods offer some significant advantages in this area owing to their ability to offer specific, selective, robust, reliable and sensitive data.
The evolution in MS technologies has resulted in significant improvements of the ion source design, sensitivity, resolution, mass accuracy and ease of use. A mass spectrometer contains three main elements – an ion source, an analyser and a detector.
The ion source is what makes the analytes of interest ionize into gaseous form in order to be transmitted through the mass spectrometer. The analyser separates analyte ions based on their mass-to-charge ratio before detection. There are different MS analysers and some of the popular types are quadrupole mass filters, quadrupole ion traps, electrostatic orbital ion traps (such as the Thermo Scientific™ Orbitrap™ analyser), magnetic sector analysers, time-of-flight analysers, and Fourier Transform ion cyclotron resonance analysers. Each of these analysers offers different operating characteristics, such as speed, resolution, mass accuracy, sensitivity and the ability to perform multiple stages of ion isolation and fragmentation, which render them well-suited to particular applications and environments. Last but not least, there are two common detectors: electron multipliers, which detect electrical charge and amplify the resultant signals to produce a spectrum; and image current detectors (used primarily with electrostatic ion traps), which produce a transient signal having frequencies corresponding to the rates at which ions oscillate.
One other important feature of mass spectrometers is the ability to controllably fragment molecules into product ions to enhance selectivity and ultimately provide confident identification. There are multiple fragmentation methods such as collision-induced dissociation, electron capture dissociation, electron transfer dissociation, and ultra-violet photodissociation.
Mass spectrometers are usually coupled with analytical instruments that either separate analytes by liquid [high-performance liquid chromatography (HPLC)] or gas [gas chromatography (GC)] phases. Chromatography is a very important analytical technique because the instrument separates the analytes of interest from the matrix based on hydrophobicities and/or polarities. Back in 1968, Viktor Tal’roze made the first attempt to interface the two apparently incompatible techniques (LC and MS). The publication in the Russian Journal of Physical Chemistry explained the spraying of a very small amount of liquid into a conventional high voltage electron impact mass spectrometer. In the present world LC-MS is considered as a common tool and continues to gain importance for detection and quantitation of a wide range of complicated analytes in complex biological matrices.
In recent decades a number of technological advances have enabled the appli-cation of LC-MS to biomolecules. What have some of these advances been and how have they benefited research into biological samples?
Since its inception about 100 years ago, MS has achieved the status of being considered as a ‘ubiquitous’ tool for research, and continues to grow in importance for applied laboratory environments. Some of the critical scientific breakthroughs with MS include discovery of isotopes, exact determination of atomic weights, characterization of new elements, quantitative gas analysis, stable isotope labelling, fast identification of trace pollutants and drugs, and characterization of molecular structure. The analytical world witnessed some significant improvement and evolution of varied MS technologies, ranging from triple quadrupole MS (QqQ) to time-of-flight (TOF)-MS to matrix-assisted laser desorption/ionization (MALDI) techniques. However, it can perhaps be said with confidence, that the importance of MS increased significantly with the invention of the Thermo Scientific™ Orbitrap™ electrostatic orbital ion trap technology, which provides true high-resolution and accurate mass analysis of molecules. The supreme selectivity of this technology allows instruments to be able to differentiate analytes from endogenous interferences (not isomers) in biological matrices. As a case in point, with an ever-increasing number of diabetes cases globally, the need for comprehensive monitoring, identification and quantitation of insulin in biological fluids has been expanding. In particular, there is a strong need for multiple analogues of insulin to be detected/ monitored/quantified. The disulfide bond in the insulin molecule makes it difficult to fragment the intact protein; however, the power of Orbitrap technology allows detection of low limits of the intact protein without fragmentation. Another example highlighting the benefits of Orbitrap technology is the ability to screen and quantify large toxicology panels. The number of designer drugs and drugs of abuse continues to grow all around the world. Toxicologists (from clinical research to forensic laboratories) look for one LC-MS assay that can detect and quantify their entire panel of drugs. However, these lists of drugs (or analytes) do not necessarily ionize in the same mode – while some ionize only in the positive mode, others ionize only in negative mode. The ability of mass spectrometers to seamlessly switch between positive and negative electrospray modes offered several advantages including the ability to easily analyse large panels of drugs that have different ionization characteristics.
QqQ technology has been in existence for a long time; however, the recent developments witnessed in the latest generation of QqQs resulted in significant improvements of speed, sensitivity, resolution [although QqQs offer unit mass resolution, and hence cannot and should not be compared to the resolution obtained from high-resolution accurate mass (HRAM) spectrometers] and robustness. While the HRAM spectrometer powered by the Orbitrap technology ensures well resolved data enabling confident identification of every single analyte in a complicated mixture, the QqQs can conduct fast, sensitive targeted quantitation of analytes. Once the target is known, it is much easier to identify and quantify the specific analyte (target) across hundreds of samples in the shortest possible time. Over the years, QqQs have become the ‘go to’ instrument for most analytical laboratories that use LC-MS as a quantitative analytical instrument.
HPLC [or ultra-high-performance chromatography (UHPLC)] plays a crucial role in the achievement of superior data quality capitalizing on LC-MS technology. Chromatographic advances in <2 μm particle columns allow UHPLC by offering higher resolution in separation. In addition, the ability of the UHPLCs to perform under high pressure increases the throughput, which in turn allows for shorter LC/MS run times. For laboratories seeking higher throughput or productivity, multichannel LCs can offer two (or more) independent LC channels, which can perform in parallel (or simultaneously) maximizing the potential of the MS. Several core laboratories have implemented this technology to run very short methods for critical and urgently required clinical research assays. Another major advancement in the world of chromatographic separation is the ability to perform online sample preparation. Online sample preparation allows the user to automatically inject the sample into the LC after sample preparation, thereby reducing the chances of any errors owing to manual transfer of samples. In addition, online sample preparation enables higher throughput compared to conventional off-line sample preparation processes. Systems that offer online sample preparation followed by LC-MS can be used efficiently for isolation of protein from the matrix, resulting in excellent separation between analyte(s) of interest from the complex biological matrices.
The world of LC-MS continues to enable advancements in technology, resulting in increased efficiency and ease of use for the clinical laboratories. PaperSpray technology with MS (HRAM or QqQs) can offer the unique ability to directly analyse samples from a plate/ cartridge where the sample is loaded as a spot. This technology not only simplifies sample accumulation and preparation steps, but also focuses on ionization of just the analyte of interest.
With very little sample volume to work with, one can remove the concern about having matrix interference and, hence, the need to use an LC, and can capitalize on the MS-based separation technique. Some critical applications that have been demonstrated using the PaperSpray technology with QqQs (in particular) comprise detection and quantitation of whole blood assay, drugs of abuse, etc.
While PaperSpray ionization can enable fast MS-based analysis, it being a direct analysis technique with no chromatographic separation and minimal sample clean-up, it can result in MS signals with high background, which may or may not affect limits of quantitation due to the signal-to-noise (S/N) ratio. Field asymmetric ion mobility spectrometry (FAIMS) is a technique that enhances selectivity of an analytical method by adding an additional dimension of separation based on mobility. FAIMS operates by applying an asymmetric waveform between a set of electrodes alternating between high and low field strengths impacting mobility of ions through a carrier gas. By applying an optimized compensation voltage, target ions pass through the electrodes, while ions not of analytical interest are neutralized on the electrode walls. By combining PaperSpray ionization with FAIMS technology, the background noise can be reduced, resulting in enhancement of the signal-to-noise ratio. An excellent example of this combination can be seen in rapid quantitation of immunosuppressants in whole blood for clinical research.
Disclaimer: The devices discussed above are for research use only and not for use in diagnostic procedures.
What are some examples of assays where MS has taken over from another type of assay?
In the world of clinical research, the advent of LC-MS has significantly changed the landscape of many assays. From endocrinology to immunosuppressants, LC-MS is used frequently by most clinical research laboratories for robust, reliable, fast, and sensitive data acquisition.
Newly developed drugs of abuse including psychoactive drugs, and synthetic cannabinoids, are often undetectable by older techniques such as immunoassays, but can be detected and measured by MS analysis. LC-MS has also been extensively used across many sporting events (human and/or animals) for detection of using illegal performance enhancers.
Biomarker research has changed with the combination of sample preparation techniques such as immunoaffinity enrichment for thyroglobulin which allows the detection of just thyroglobulin and not anti-TG. For studying cardiovascular disease, apolipoprotein quantification can be routinely detected in QqQ instruments compared to physicochemical fractionation. Catecholamines and metanephrines are analysed by radio immunoassays (RIAs) and enzyme-linked immunosorbent assay (ELISA). While ELISAs offer a simple method of analysis, LC-MS with mixed-mode columns can offer better separation between tricky analytes that tend to coelute (such as bound and free metanephrines). Such advantages are also allowing increased adoption of LC-MS for biomarker research.
Can you provide some examples of diagnostic assays that are in the pipeline for routine lab use?
LC-MS as a technology finds a wide variety of use in every clinical research laboratory – addressing identification, confirmation and quantitation needs for small to large molecules. Needless to mention that a technology of such power can be used across a host of disciplines. While analysis of vitamin D, steroids, hormonal drugs, immunosuppressants are considered as routine in today’s world, LC-MS (with either HRAM or QqQ) is being used for disease detection and therapeutic regime optimization for several other disease types. These include cancer, endocrine disorders, Alzheimer’s disease, infectious disease detection and monitoring, as well as newborn screening for conditions such as hemoglobinopathy and lysosomal storage disorder.
The assays described in this section are for clinical research only, and not for diagnostic use. In the world of precision medicine (or clinical translational research), there is an increasing need to combine knowledge from discovery to quantitation in proteomics, lipidomics and metabolomics. The discovery workstream transforms to quantitation once targets are determined. The quantitative assays are usually performed on QqQs.
The world of toxicology can benefit from implementation of LC-MS technology to address the continuously increasing challenge of more drugs of abuse molecules being added to their list for targeted screening and quantitation. LC-MS technology along with an extensive library of molecules represent proven methods that can ensure that every toxicology laboratory (from clinical research to forensic toxicology laboratories) achieves superior confidence in data, regardless of the number of analytes they have to monitor.
Clinical research laboratories can also benefit significantly from other innovative technologies such as the PaperSpray ion source for MS, which employs new ionization strategies to decrease the amount of sample preparation by directly ionizing components of a sample deposited on paper, with data acquisition typically being completed in under two minutes. The cartridges used in this technology allow for easy sample collection and storage as well, which will be important in pathology and clinical chemistry. In addition, ion mobility MS may be employed for gas-phase separation of analyte ions based on a combination of factors, such as charge state, shape, conformation and size. This has been important in improving sensitivity and selectivity in clinical assays.
Currently most MS assays used in clinical labs are laboratory developed tests (LDTs), i.e. developed by experts in MS for use in that lab. What are the challenges that have to be overcome or what technological developments need to take place before MS-based assays can become ‘black box’ in vitro diagnostics (IVD) tests?
LC-MS represents a complementary technology to conventional chemistry and immunoassay-based techniques and offers greater specificity, speed, analyte range, throughput and multiplexing capabilities coupled with a lower cost per sample and reduced sample volumes. Performing LDTs by LC-MS enables clinical diagnostic laboratories to replace expensive chemistry or immunoassays run on random-access automated lines with more economic batch testing. Positive immunoassay screens are typically followed by LC-MS confirmation and quantitation. But where positive rates are high, switching solely to identification and quantitation by LC-MS often makes economic and operational sense. However, there are some other criteria that are expected when it comes to adaptability of LC-MS in the applied laboratories. The process to convert LC-MS to a ‘box’ that can offer critical answers with the same efficiency as obtained in the clinical research laboratories can be complex. The traditional LC-MS systems can be referred to as ‘open systems’ owing to the fact that the LC and MS are not in one box. The benefits of open systems lie in the ability to customize their functionalities, along with the ability to optimize and change sample preparation protocols. A specific combination can be created for a specific assay; however, it might be challenging to ensure the ability of such boxes to conduct multiple assays, as is typically done with LC-MS systems in the lab. Even for a specific assay, the LC-MS system will need a comprehensive process along with strict protocols and methods that can be operated by the push of a button. In the last couple of years, the world of LC-MS has witnessed evolution of clinical analysers with LC-MS that can offer sensitive, robust analysis of analytes such as vitamin D and immunosuppressants. Although it can be considered as early days, clearly the evolution of LC-MS as clinical analysers indicates a transition in the right direction.
What do you envisage for the future of MS in clinical diagnostics?
In the world of clinical research, fast evolution of LC-MS technology, a good understanding of government regulations and the expectations of every laboratory (from achieving confident results to lowering their cost/sample or cost/analysis) will make MS a prominent technology in clinical research. Automation in sample processing and handling may help the integration of MS into everyday use.
In the world of clinical translational research, expanding proteomics, lipidomics and metabolomics to genomics and transcriptomics will give a holistic perspective of biological pathways that can enable discovery of novel therapeutics for various diseases. In alignment to the progress observed in next-generation and whole-genome sequencing, MS and bioinformatics are opening new domains with their ability to offer complementary data enabling the world of precision medicine.
Metabolomic and proteomic technologies comprise several MS applications, both with and without LC or GC. In addition, the use of MALDI-TOF can offer rapid, automated analysis and as a final step in the process, powerful informatics systems have set the standard for new MS clinical applications. As expected, the MS instruments and the assays developed on them will deliver faster turnaround times and increased sensitivity and specificity, which in turn will ensure clinical research laboratories obtain improved results with optimal combination of automation. Online automation will use cloud computing to enable result and data harmonization as well as data portability.
MS and its use in clinical research laboratories have experienced significant technological advancements in the last couple of decades. However, there’s a lot more that can be achieved with this technology, and continuous engagement between the clinical research and MS manufacturers can enable the increased uptake of MS for everyday use in a clinical research laboratory.
Debadeep Bhattacharyya PhD Senior Manager
Clinical Research and Toxicology
Thermo Fisher Scientific, Boston, MA, USA