May 15, 2018 (Vol. 38, No. 10)

MaryAnn Labant

A Few Modifications Might Enhance Performance

Even if your trusty old mass spectrometer is firing on all cylinders, it may seem underpowered, particularly if you’re trying to run down impurities, degradants, elemental markers, metabolites, and other elusive species. You may even feel as if your better equipped colleagues are preparing to pass you by.

Don’t despair. Instead, find out what the leading mass spectrometry (MS) mechanics are doing to boost the resolving power of their machines. After a little time under the hood, you may decide that you’re ready to shift your basic research, drug development, or quality control projects into high gear.

Recent innovations include isotopically calibrated spectral analysis software, instrumentation to detect metals within individual cells, and methods to label the entire metabolome to facilitate inter-laboratory comparisons. These innovations, and many others, will be presented at the 66th American Society for Mass Spectrometry (ASMS) Conference on Mass Spectrometry and Allied Topics, which will take place June 3–7 in San Diego. In the meantime, we invite you to watch some powerful MS systems as they take a few qualifying laps in this feature article.

Finding Impurities and Degradants

High-resolution instruments provide formula identification through accurate mass analysis. However, in routine quality control of pharmaceuticals, it would be more economical and practical if the identification of unknowns could also be accomplished on a more common, cost-effective instrument, such as a single quadrupole LC–MS instrument. In their usual role as chromatography detectors, single quadrupole LC–MS instruments deliver nominal mass or unit mass resolution. They do not provide accurate mass analysis.

An MS measurement is not a single line for a given ion but a unique isotopic fingerprint whose pattern is unique for every formula. But the imperfect “electromagnetic lens” of an MS instrument produces imperfect line shapes. Ion measurements taken at various times on the same or different instruments will not yield, upon analysis, identical fingerprints.

Fortunately, if they receive the right enhancements, general-purpose instruments can provide reproducible, accurate mass data and formula identification. One such enhancement is Cerno Bioscience’s novel calibration technology, a software solution that can significantly enhance mass accuracy (typically 100×), enabling accurate mass measurements for formula identification.

In addition to giving accurate mass information, the software evaluates the isotope fingerprint to show the best match between it and calculated formula profiles to further enhance formula identification through “Spectral Accuracy.”

This process is incorporated into CLIPS (calibrated line-shape isotope profile search), a key technology incorporated into the software. After the accurate mass confidence level is determined for a mass peak, the candidate formulas are further analyzed for Spectral Accuracy, reducing the possible candidates from 30 to 40, for example, to perhaps three or four.

The software enhances the discriminating power of routine MS instruments, bringing identification capability downstream to the quality control (QC) technician, reducing costs, accelerating the process, and improving the QC process overall.

“Our software and algorithms enable formula identification on the standard QC instrument,” asserts Don Kuehl, Ph.D., vice president of marketing and product development at Cerno Bioscience. “[Our technology] provides a much higher level of confidence for identification of impurities and degradants in [chromatographically] well separated samples.

“In addition to calibrating for the mass position, our software also calibrates the line shape. We can then calculate the theoretical isotope pattern to be the same and exactly perfect every time. This is an extremely powerful dimension to further qualify the identification of an unknown compound.”

Cerno Biosciences provides a technology for formula identification called calibrated line-shape isotope profile search (CLIPS). Unlike approaches to elemental composition that use only mass accuracy for formula identification, CLIPS adds another dimension by matching the full isotope profile of the unknown to the theoretical profile. In the past, this has not been possible due to the uncalibrated line shape in mass spectrometry.

Single-Cell Analysis

Another extended capability for quadruple-based technology is single-cell analysis. Traditional metal content measurements use nominal mass concentrations, which assume an equal distribution of metals among the cells in a cell population, overlooking vital information on metal distribution and variation on a unicellular level.

Introduced in March 2017, the PerkinElmer Single Cell ICP-MS (SC-ICP-MS) is an automated technology capable of rapidly measuring and quantifying the metal content of live or fixed individual cells down to the attogram-per-cell level, the metal mass distribution, and the number of cells containing the metal. SC-ICP-MS can scan for any metal in the periodic table in cells ranging from 0.2 to 100 µm in size.

Accurately quantifying the metal content in an individual cell can offer insight into the uptake and elimination mechanisms of metals and/or metal-containing nanoparticles; the mechanisms of interactions between metal-containing drugs and cells; and the distribution of micronutrients, such as zinc, copper, or iron.

Hardware includes one of PerkinElmer’s NexION family of ICP-MS instruments (capable of acquiring 6 million data points/min at 10-µsec dwell time), a PerkinElmer Asperon spray chamber (capable of delivering individual, intact cells into the plasma of the ICP-MS), and a microfluidic workstation. Any shape or size well plate can be used, and sample volumes can be as low as 5 µL. The company’s Syngistix Single Cell Application Module can be used to perform data collection and processing.

The technology has been used to determine the uptake and interaction of toxic metals in bacteria and plant cells, the bioavailability and bioaccumulation of gold particles or ionic gold by algae, the efficiency and effectiveness of drug delivery monitoring cisplatin uptake in ovarian cancer cell, and the usefulness of single-cell approaches in nanomedicine.

“Gold nanoparticles (AuNPs) are showing emerging use in biomedicine for cancer diagnosis and therapy,” discussed Chady Stephan, Ph.D., senior lead inorganic applications, PerkinElmer. “However, the quantification of AuNPs uptake into cancer cells using elemental analysis has been limited to ensemble measurements, which require digestion of the entire cell population in concentrated acids.

“This approach assumes that all cells within a given cell population interact with the same number of AuNPs. To test whether this assumption is correct, new analytical strategies were needed that allow quantitative elemental analysis at a single-cell level.”

Isotopic Ratio Outlier Analysis

In metabolomics, interlaboratory result comparisons present an analytic challenge because they must allow for varied methodologies and limited reference standards. Also, they cannot readily adapt an approach that is often used in classical targeted quantification assays, an approach that provides relative quantifications based on the relation of the sample to purchased isotopic standards. In the metabolomics context, this process is restrictive and costly because individual analytes require corresponding standards, which may not be available for all metabolites.

Isotopic ratio outlier analysis (IROA) allows for the generation of isotopically labeled control standards of an entire metabolome by growing cells in 95% randomly 13C-labeled glucose. Naturally occurring isotopes are positive; IROA reverses that and creates a negative effect of n−1 isotopes.

The IROA-labeled cells are spiked into the sample. Since the spiked-in amount is known, quantitation for each metabolite can be performed through a ratiometric approach. This allows pattern comparisons from multiple methods and laboratories, and multiple cell lines can be grown with the media.

Designed to provide cost savings, IROA makes it easy to find the labeled species and allows untargeted discovery of metabolites that have not yet been curated. A key aspect is that only enzymatic metabolites get labeled, helping to discriminate noise and artifacts.

“We employed IROA to help other people learn how to use it,” states Timothy J. Garrett, Ph.D., associate professor of pathology at the University of Florida and co-director of the high-throughput MS metabolomics core of the Southeast Center for Integrated Metabolomics. “We developed standard operating procedures and improved the software to ensure that it is looking for and picking things correctly. We are also testing to make sure the technique is applicable to human studies.”

The technique was initially tested in yeast extracts, then it was tested with human-based samples, where it was found to work well. Next steps include evaluating human cell lines, developing the quantitative methods, and scaling up for clinical diagnostics. Reference ranges will be developed and tested to determine correlation with ranges in the clinical setting as well as approaches defined to identify unknowns.

“Metabolism is the closest to phenotype,” insists Dr. Garrett. “Metabolomics is gearing up to discover new metabolic associations in known and rare diseases to help improve the way we diagnose diseases.

“If we can then pull out and understand all the metabolic information, it will help us develop better diagnostic paradigms. Years ago, this is what newborn screening did, it transformed the way we diagnosed newborns.”

Isotopic ratio outlier analysis (IROA) was used by University of Florida researchers to generate this tryptophan analysis, which shows how the 95% 13C-labeled sample provides an n-1 isotope pattern starting at 216.1345. Peaks for the labeled sample (purple shading) are compared to peaks for the unlabeled standard (tan shading).

Coping with Higher Pressures

Samples generated by omics and environmental samples may contain more than 10,000 compounds. Such complex mixtures can overwhelm current liquid chromatography (LC)–MS systems, which lack sufficient resolving power. Although these systems can achieve higher resolving power if they make use of smaller resin particles and longer columns, both these changes require that the systems operate at higher pressure.

Commercial systems are currently limited to operating between 8,000 and 20,000 psi, depending on column dimensions. To see if this operational ceiling could be raised, Robert Kennedy, Ph.D., Hobart H. Willard Distinguished University Professor, professor of chemistry, and professor of pharmacology at the University of Michigan, experimented with modifications to a proprietary ultra-high-pressure liquid chromatography (UHPLC) system.

The UHPLC system capable of operating at inlet pressures of up to 40,000 psi and was coupled online to a high-resolution mass spectrometer. The technical limit of pressure was addressed through a special arrangement of pumps and valves. Existing technology has allowed pumps to operate as high as 100,000 psi; however, this technology is not suitable for gradients.

A gradient pump was used to form a gradient at low pressures, and then the stored gradient was driven using the very high pressure pump. Certain key components, valves and fittings that can withstand the high pressures, were invented by a collaborator.

The system has been used for initial test analyses of metabolomic and lipidomic samples. According to Dr. Kennedy, it is producing clearly sharper peaks, which in turn should mean more resolved peaks, as compared to a commercial capillary LC system. It remains to be determined if the system affects the information output of an undirected metabolomics experiment.

More study is needed. A lot remains unknown about packing columns to get the theoretical efficiencies possible with high pressures and finding the sweet spot for column and particle dimensions. Although efficiencies may be predicted with theory, they depend on how well the columns are packed.

In addition, the plumbing of the system is not yet ideal for compounds that are not well retained. The dead-time peaks can be broad; further investigation is required to see if this complication can be eliminated. Dr. Kennedy also plans to test more complex mixtures of peptides and proteins to see if the proteomics will be improved.

Developed by scientists at the University of Michigan, the UHPLC system shown in this image can operate at pressures as high as 40,000 psi. The gradient pump (upper center) creates a mobile phase gradient and delivers it to the gradient loop (bottom left). The pneumatic amplifier pump (bottom center) then drives the fluid into the column (upper left) at high pressures. Flow is controlled by freeze-thaw valves (bottom left). The column is interfaced to a quadrupole time-of-flight MS (right).

Previous articleCommon Cold Blocked from Replicating by Novel Drug Molecule
Next articleNDF Identified as Critical Molecule for Facilitating Transcription through Nucleosomes