Mass Spectrometry Solutions for Biology on Display at ASMS

The 70^th annual meeting of the American Society for Mass Spectrometry (ASMS) was held last month in Minneapolis. With mass spectrometry (MS) growing rapidly—and a top-down analytics forecast of over 17,000 MS instruments to be installed globally in 2022—the meeting qualifies as one of the most important in the science space.

The installed base has been largely driven by advances in the applications of MS. Rapid advances in instrumentation contribute to the short product life cycle (~ 5 years), leading to a significant replacement market.

‘Omics

MS already enjoys a technology life cycle spanning more than 70 years. The first 50 were focused development of the mass analyzer technology, but the advances in the last 25 have been in applications. Instrument development has responded to the market need for solutions. For example, a few years ago, leaders in ‘omics started to anticipate single cell analysis. They noted that the measurements of large cell populations were not very effective, since the key events in disease, especially in the early stages, involved only a few, perhaps only one, cell(s). The signal from the infectious event was swamped out by the gigantic background from the normal population. The leaders anticipated needing to study single cell heterogeneity and correlate this with ‘omics events and disease for drug research and ultimately human diagnostics. Coupled drugs and diagnostics is the vision of precision medicine.

Traditional approaches that involved samples consisting of many cells failed to see critical changes since the background of normal cells was so high. In particular, the key event leading to disease was not observable until the disease had progressed to many multiple cells. Cancer therapy is much more effective if the mutation is caught at stage 1, rather than 4.

CellenlONE from Cellion is a key platform for multi ‘omics of single cells. It selects, images and dispenses single cells into 384 or 1536 well plates or other formats. The cell suspension slowly recirculates through a flow viewing station. Only when the view field shows a single cell is the piezoelectric dispense function activated and the solution containing the lone cell dispensed. The image of each cell is retained for reference. The CellenONE automates an operation that for humans would be very tedious.

Multiplexing assays for LC-MS analysis is one way to increase throughput. Thermo Fisher Scientific (TFS) extended its line of Tandem Mass Tags for multiplexing to 18. Previously, TFS had previously offered 6, 10, 11, and 16 plex kits. Each kit member couples its multiplex tag through amine groups on protein digest peptides. The reagents are isobaric products but with a unique MRM pattern in MS/MS.

Targeted assays for ‘omics and clinical diagnostics may be able to directly analyze samples without a separation unit as the front end. Covaris presented Adaptive Focused Acoustics for processing tissue specimens, such as FFPE, quickly prior to analysis by MS alone. Covaris’ approach is to focus ultrasound on a multiwell plate of specimens to disrupt the samples, such as tumors. Simultaneous processing of all wells in the plate is followed by sequential processing of each well, often with a cycle time of a few seconds/well.

Clinical Dx

Mass spectrometry has been on the borderline of acceptability for in vitro diagnostics for years. Add in an HPLC, and the border fence gets even higher, especially in the U.S. This has led to classifications of “Research Use Only” (RUO) and Laboratory Developed Tests (LDT), and “for IVD” etc. These regulatory high hurdles protect the public, but carry a large economic burden. For example, American diagnostic labs may need backup instruments to maintain operation during down time of the first MS. Lab staff may need redundant skills and training to maintain productivity in case of sickness, vacation or turnover.

Waters has addressed this issue with introduction of three LC-MS instruments specifically targeted for in vitro diagnostics (IVD)—Xevo TQ-S Micro System for IVD, XEVO TQ-XS IVD System, and Xevo TQD-XS IVD. All are supported by an IVD field organization designed to meet the support requirements for the labs, plus products designed to ensure high data quality. Maintaining very high reliability is a key metric of both the vendor, customer and patient.

Waters representatives cited a dramatic IVD report during ASMS: according to third party research, 82% of organizations surveyed experienced at least one unplanned outage involving critical assets over the past three years, leading to lost production costs averaging $260,000 per hour. This illustrates concern about the expense of down time. Accordingly, Waters has introduced FlexCHOICE, which is a customized field service program designed to meet the unique requirements of a lab, such as two-hour call back, etc. Anticipated customers include regulated labs in IVD, pharmaceutical, contract services and foods.

Mass photometrics in gene therapy

One of the most interesting approaches to gene therapy is to encapsulate genetic material in a particle, such as a virus shell. The idea is to use the virus shell to deliver genetic material to sick cells using the virus’ infection machinery. One of many key questions is, how many of the virus constructs actually carry the drug active?

A report from the Wysocki lab at Ohio State University described the use of SID-CDMS for measuring the partial filling of the AAV capsid. SID greatly simplifies electrospray ionization by reducing adduct formation. Adding SID (Surface Induced Dissociation), which is very mild, provides more detailed structural information on the drug product. 

Refeyn has introduced the SamuxMP mass photometer, which can provide an assay of % full and empty with μL samples in about 5 minutes. One example is the empty/full ratio for adeno-associated virus. The company says photometric measurements do not require as sophisticated instruments as a mega Dalton MS. Wyatt Technologies and Tosoh BioSciences, for two, probably agree.

Portable instruments

Baspec showed the latest model of their transportable mass spectrometers, which uses a miniature linear ion trap that provides MS/MS capability. The Continuity provides higher detection sensitivity for trace detection. The Portability MS is smaller-designed for more routine needs. Ion sources include electron impact, (EI), thermal desorption (TD), electrospray (ESI), atmospheric pressure chemical ionization (APCI), DART and DESI. Designed for at-site applications, the instrument should appeal for food safety, pathogen detection, controlled substances, and chemical warfare and security applications.  

Direct Mass Spectrometry with Water’s Radian ASAP seems to offer so much more than detection of marker compounds or unexpected contaminants. The Radian ASAP is based upon Waters’ ACQUITY QDA Mass Detector with a direct injection Atmospheric Solids Analysis Probe (ASAP), which provides APCI MS. APCI gives good spectra for thermally stable, volatile, mid polar, analytes with m/z less than 1000 Da. Samples are loaded into disposable glass capillaries that are then loaded into a horizontal direct injection device. Applications include confirming label content of foods, beverages, and oils and monitoring chemical synthesis and purification. The tag line is “The Power of Knowing.” I expect that Radians will soon be a community instrument, especially in core facilities.

GC-MS

GC-MS is a work horse in many labs, with a market value of about $2 billion. But there is a problem: helium is the carrier gas in most installations. Helium is getting so scarce and hard to obtain that many chromatography labs are considering switching to hydrogen for carrier gas. However, some MS instruments are not designed to tolerate hydrogen. Hydrogen in the vacuum pump oil can reduce pump performance.

LECO points out that the Pegasus BT is designed for H2 carrier gas. Another potential problem is chromatography data systems might not be able to keep up with the high speed obtained with low viscosity H2 carrier gas. They cite an example of a 16-minute separation optimized with He, but upon switching to H2, the separation required only 3.45 minutes. This will require updating the retention time file and may be too fast for some legacy chromatography data systems.

About a decade ago, Jaap De Zeeuw of Restek presented an astounding poster showing how to triple the throughput of GC-MS by using the vacuum system of the MS to reduce the pressure in the GC column. It seems counter intuitive, but is not. The technology did not attract much of a following. However, in 2022, Restek introduced a kit for Low Pressure GC-MS (LPGC-MS). By using a 0.53 mm analytical column that is inserted directly into the MS and a flow restrictor on the GC inlet side, one can benefit from low pressure created inside the 0.53 mm column. Using this LPGC-MS technique, some efficiency is traded for speed. But, because a mass spectrometer is used, most coeluting components can be deconvoluted by the MS. One study of a complex mixture of pesticide residue standards produced peak widths of 1.5 to 2.0 seconds. The 1 μm film increases retention and peak capacity.

This design enables the chromatographer to use the analytical column near its optimum linear velocity for maximum resolution. Lowering the operating pressure in the column decreases the carrier gas viscosity, which increases the optimal linear velocity significantly. Restek’s LPGC-MS column kit relies upon a low-thermal mass connector between the chromatographic column and deactivated restrictor capillary, and also the transfer line. Leak free performance has been demonstrated for 500 thermal cycles between 70 and 320^oC. If this kit is a viable solution in customer’s hands, I expect Restek will provide a wider selection of stationary phases for the chromatographic section.

LC-MS/MS

Table 1 forecasts that LC-MS/MS will place 4,000 units in 2022. These generally carry a price tag of over $100K. Thus, laboratories are investing over $4 billion for these instruments. The reason is tandem mass specs provide the data that supports decisions especially when health and safety are involved. Here are some examples.

Mycin drugs (Aminoglycosides) are economical antibiotics that may be found in meats, milk and eggs. However, they can bioaccumulate with high avidity leading to potential oto- and nephron-toxicity in humans. Regulatory limits for withholding periods and residue levels are established. Shimadzu has developed an HILIC (Hydrophobic Interaction LC) assay with a triple quadrupole MS with a 4.5 min cycle time. When required, confirmatory runs use a 15-minute cycle time plus 15 MRM/compound.

Most mass spectrometers use an electric field to guide ions from the sample source (LC column) to the mass analyzer. Imperfections including dirt and residue from previous samples distort the flow profile by surface charging. Hence the need for too frequent cleaning of the ion source. PerkinElmer introduced a triple stage Hot Surface Ion Desolvation inlet that desolvates the ions with a controlled vacuum gradient. Ions are transported by expansion of a flux gas through each stage in a laminar manner. This minimizes the peak broadening effects from electrostatics, which improves the ion profile before the first stage in the mass analyzer. With the laminar Flow Ion Guide, detection stability is improved, while extending the uptime of the LC-MS-MS. The Ion Guide is designed for PE’s QSight LC-MS/MS.

Decimal shift in limit of detection

Perkin Elmer’s NexION 5000 ICP-MS has added a fourth quadrupole stage to the MS detector.  The additional stage enables isolation and focusing of ions while ignoring photons and ions with non-target analytes. However, the big advantage of the fourth quad is to add a reaction cell where ions can interact with added reaction gases (O2, NH3, and CH4) to form adducts that move the mass of the target ion to a higher mass region of the spectrum. This reduces interferences and may also have significantly lower background noise. Generally, the fourth quadrupole of the NexION improves LOD for elemental analytes by a factor of 10. However, if interferences are a problem, then removing them via adduct formation can yield a larger improvement.

Analysis of polymeric materials

Polymers are everywhere. Their chemistry is very complex. Solution-based analytical tools are often not suitable for polymeric solids, especially if one is interested in surface chemistry. Bruker says MALDI MS may be a very useful tool.

Some examples: Reactive groups are often grafted to polymer ends to provide active sites for coupling, in block copolymers. This chemistry needs to be studied, and the reaction products characterized. The MALDI fragmentation patterns provide primary structure, sequence and chemical topology. This is particularly useful in defect analysis where the local chemistry may be quite different than the bulk structure. With time, the plastic surface can be exposed to a variety of chemicals, which can produce a range of agglomerants. MALDI is one technology that can quickly bridge the gap between physical and chemical structure of microplastics.

But the polymers may be very large … in the mega Da range, which can be a challenge for SEC and field flow fractionation. But MALDI uses the high mass discrimination power and huge dynamic range to follow the grafting reaction. The fragmentation patterns provide primary structure, sequence, and topology. MS is very useful in identification of minor components that may phase separate. MALDI imaging is already used for imaging of biological systems—extending the imaging to non-biological polymers and structures is a natural extension. The microstructure of polymeric materials is pertinent to their bulk behavior and degradation.

Polymer defects are a natural target for MALDI since the sample can be examined without liquification. Some plastics are specifically designed to be insoluble.

Ion mobility

During the last decade, high-end mass spectrometers added ion mobility stages to the ion optics mass analysis train. This was particularly effective for resolving interferences from isobaric analytes, such as isomers. The ion mobility stage was usually less than 25 cm. About 5 years ago, Waters introduced the SELECT SERIES Cyclic IMS, which included their SYNAPT XS IMS ToF mass analyzer. Both are fast technologies. The cyclic flow path was very useful in resolving ions with very small differences in collisional cross section (CCS). The separation is orthogonal to chromatography, which makes it useful in untargeted assays.

At about the same time, ion mobility was also being studied by Richard Smith’s lab at Pacific Northwest National Laboratory (PNNL). He had a different approach using drift path etched into printed circuit boards. His team found it was easy to fold the drift path without losing resolution at the bends. This enabled a smaller drift chamber, which lowered cost. This year, the PNNL team showed that multiple circuit board can be stacked so the path length can be increased. The sample starts on one board then passes through a hole in the first board and on to the second, third … and so on to the TOF-MS.

New mass spectrometers

Agilent upgraded their popular triple quadrupole platform to the 6475 by adding a superheated Agilent Jet Stream (AJS) ion source, curved and tapered collision cell, heated hyperbolic quadrupoles, ± 20 kV high-energy dynode detector system, and intelligent secondary injection workflows. The latter is useful when an out of specification result (OSR) is observed, including errors due to excessive carry over, failed injection or a too concentrated sample. Such events trigger repeated injections, reducing sample volume or adding blanks.

The 6475 is compatible with several ion sources. Agilent’s VacShield design facilitates maintenance of the ion source without breaking vacuum. Source maintenance can be completed in less than 30 minutes. When coupled with an HPLC, the LC-MS instrument detection limit is 3.5 fg or ppt. Early Maintenance Feedback (EMF) monitors performance of the MS to warn the operator of impending maintenance needs. Data integrity is designed to comply with US FDA CFR Part 11, and EU Annex 11.

Agilent is responding to concerns about the product life cycle. The 6475 received an ACT Environmental Impact Factor score of 54.8—too high to be considered good. The ACT score is a measure of a product’s environmental impact, including factors such as design, manufacturing, shipping, and power consumption.

Conventional wisdom for the last three decades is that quantitative analysis requires quadrupole mass analyzers. The new Xevo G3 QToF from Waters challenges this generalization by designing a ToF mass analyzer for quantitative analysis, without sacrificing performance for quality. The Waters Xevo G3 delivers a liner dynamic range of 10^5 or more. Plus, it has the speed of a ToF. The mass range extends upward from small molecules to complex biotherapeutics.

Summary

Market and sales interest in MS, including hyphenated techniques, is very strong, particularly in healthcare-related R&D. Application needs are leading to design innovations. The market for MS systems in applied and regulated laboratories is expanding and could become very large as the firms become more flexible and creative in their support packages.