Triple Quadrupole Mass Spectrometry Advances Pharmaceutical Analysis

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For both small- and large-molecule applications, advances in mass spectrometry are leading to interest in this technique as an alternative to liquid chromatography.

Mass spectrometry (MS) is an important analytical tool for the evaluation of both small- and large-molecule pharmaceuticals. In small-molecule drug manufacture, very high sensitivity is required for the detection of impurities and other contaminants, such as extractables and leachables in parenteral formulations. Speed of the analytical technique and the uptime of the instrument are also critical. High resolution and mass accuracy are also important for the analysis of biopharmaceuticals, and MS systems are used throughout the development and optimization process. As the complexity of drug therapies increases, regulatory requirements become more stringent and many active pharmaceutical ingredients (APIs) become increasingly potent, there is a continual need for improvement of both the sensitivity and speed of analytical techniques. New mass spectrometers from Thermo Fisher Scientific address these issues through a combination of technology advances and software developments.

Current uses for MS

Mass spectrometers are typically quite expensive and thus have generally been used where alternative analytical techniques cannot provide the level of performance required. In small-molecule manufacturing, therefore, MS is used for the analysis of impurities in drug products. Detection of extremely low levels of extractables and leachables in large-volume parenteral formulations, for example, is testing the limits of conventional analytical techniques, and there is growing interest in MS for this application. It is also becoming a valuable technique for the analysis of highly potent drugs due to the very low API concentrations found in these types of products. “In these cases, triple quadrupole mass spec instruments provide the resolution, specificity, and speed that is needed,” says Patrick Bennett, marketing director for pharmaceuticals and biopharmaceuticals with Thermo Fisher Scientific.

In biopharmaceutical manufacturing, there is a need to analyze with high resolution and accurate mass very complex molecules. As a result, MS is widely used through the development, optimization, and manufacturing of large-molecule drugs. “Whether identifying which clone to use, evaluating the impact of different nutrients on production, or characterizing the protein or peptide product, high resolution, accurate mass spectrometry is used in order to correctly determine the structures and quantities of the compounds of interest in very heterogeneous and complex systems,” Bennett observes.

Need for improvements

Regardless of the application, increased resolution, mass accuracy, and speed are always desired.   “The pharmaceutical industry is evolving, and analytical techniques must develop to meet the changing environment. While a small-molecule pharmaceutical manufacturer using a triple quadrupole instrument might obtain adequate chromatographic resolution and sensitivity by pushing an older instrument to its limits, the consequences of doing so are often more challenging method development and sample analysis, and more frequent maintenance resulting from larger injection volumes. In biopharmaceutical manufacturing, given the complex systems that need to be analyzed, there is always demand for greater sensitivity and improvements in the quality and quantity of the data, and getting all of that information in less time,” explains Bennett. Outside of the performance of the instruments, ease-of-use is another aspect that can impact the utility of mass spec as an analytical technique.

Advanced triple quadrupole technology

Two new triple quadrupole instruments from Thermo Fisher Scientific (TFS) were recently introduced to address the need for increased sensitivity for small-molecule analysis. The TSQ Endura triple-stage quadrupole mass spectrometer has a new ion source design and interface. In addition, an ion-beam blocker that prevents neutral ions from contaminating components improves the robustness and dramatically increases uptime, according to Bennett. First, the ion source is self-contained, with all gas, electrical, and other connections made automatically as it is plugged in. Secondly, the system was designed to be easy to clean. No tools are required to maintain the system up to the point of the first quadrupole. “This system is ideal for workhorse applications requiring trace level quantitation, including pharmaceutical quality analysis and quality control, where long maintenance intervals are important,” Bennett notes.

The TSQ Quantiva triple-stage quadrupole mass spectrometer has the same new source and simplified maintenance design as the TSQ Endura, plus several other significant engineering changes, according to Bennett. First, active ion management (AIM) is used to optimize ion creation and transmission from the source to the detector, resulting in extreme sensitivity to the attogram level (see Figure 1). “The opening for the ions was changed to more of an oval shape, and as a result provides much more efficient solvent evaporation, and thus provides much lower chemical noise. The ion path, optical transfer line, and the collision cell were also modified,” he explains (see Figure2). The increased scanning speed of the new design also makes it possible to screen several hundreds of compounds in a single analysis.


New tribrid design

Thermo Fisher Scientific has also introduced a novel tribrid system that is the first instrument to combine three mass analyzers—quadrupole, Orbitrap, and linear ion trap – in a new architecture (see



). The Thermo Scientific Orbitrap Fusion Tribrid offers improved biomolecular characterizations and can collect twice as many scans per unit time at significantly greater sensitivity than was previously possible, according to Bennett (see



). “We have found that we get much greater proteome-wide coverage with much greater quantitative accuracy than ever before using the Orbitrap Fusion Tribrid system,” says Steven Gygi, professor of cell biology at Harvard Medical School.

In addition, Bennett notes that there are three possible fragmentation methods available that can be run in tandem or in combination in ways that were never possible before, thus making it possible to dramatically reduce the number of individual experiments that must be completed. “A typical mass spec run might last 1-2 hours. If a company needs to do 100 runs for a given product, with the Orbitrap Fusion Tribrid, that number could potentially be cut in half, saving 50-100 hours of analysis time.”

Interest from unexpected places

The new technologies were introduced in early June at the American Society for Mass Spectroscopy Conference on Mass Spectrometry and Allied Topics and have since garnered significant interest, according to Bennett. As might be expected, most of the requests for demonstrations are coming from existing customers or new customers looking to use the new instruments in expected applications. Bennett comments that the Orbitrap Fusion Tribrid, which would typically find use in biopharmaceutical analysis applications, is also attracting the attention of people who would normally use a triple quadrupole system for quantitative analysis. “There seems to be growing recognition that the ability to use three different fragmentation methods with three types of analyzers to obtain very high resolution results very rapidly would be, despite the higher price of the tribrid system, highly beneficial for metabolic studies for large molecules as well as other quantitative analyses,” Bennett observes.

Making mass spec more accessible

Making all of the new systems easy to use was a prime goal for Thermo Fisher Scientific. The user interface is simple with click-and-drag operations. All of the systems also include new software that provides templates for rapid method development and data acquisition. There are also special software programs designed to assist with protein and small-molecule identification  as well as mass spectral databases for the identification and structural elucidation of unknowns.

“Our goal was to develop highly sophisticated systems that provide the resolution, speed, and mass accuracy needed for various pharmaceutical industry analyses, but make them easy to use by both those with extensive training and those that are new to mass spectrometry. Regardless of their backgrounds, researchers, and operators do not have time to spend preparing to run experiments. They need to be spending that time reviewing the results and making decisions,” states Bennett.

Transformations to come

Mass spectrometry is making advances in adoption with the pharmaceutical industry. “We are right at the initial stages of market adoption in pharmaceutical manufacturing. With continued technology evolution, we will see growing use of MS analysis throughout the development, optimization, production, and downstream applications in pharmaceutical manufacturing.”

Bennett even sees mass spectrometry having a role in process analytical technology (PAT) applications in the future. “We are working with partners to develop fully automated systems that can take advantage of the rapid analysis times and high sensitivity that mass spec offers.” He notes that PAT systems for small molecules do not present as much of a challenge as those for large molecules because biopharmaceuticals require a digestion step that must also be automated in some way.