Mass spectrometry (MS) is an analytical technique for measuring the mass-to-charge ratios (m/z) of molecular and atomic species. In essence, MS determines the most intrinsic property of a molecule or atom: its molecular or atomic weight. Prior to 1970, mass spectrometers were large, expensive instruments with very high power demands and very low reliability. Quadrupole mass filters changed everything and allowed modest MS units that focused on environmental applications to gain in popularity, so environmental monitoring was in full fashion for a while. These instruments were refrigerator-sized units that were accompanied by minicomputers of similar dimensions. But their mass resolution was not much better than one atomic mass unit, and there were no tandem MS (i.e., MS/MS) units at that time. Gas chromatography-mass spectrometry (GC-MS) eventually became the tool of choice.

Meanwhile, traditional mass spectrometers gained use in biomedical applications, particularly in characterizing inborn errors of lipid metabolism. Leading biomedical mass-spectrometry laboratories conducted studies of complex mixtures such as steroids and prostaglandins. Studies of small polar molecules such as neurotransmitters that were made volatile for GC-MS with various reagents also received a lot of attention. Inevitably, the less costly quadrupole instruments began to be explored in biomedicine, and once the triple quadrupole system for MS/MS became widely available in the early 1980s, every medical school wanted one.

Until the 1990s, there was no practical coupling of liquid chromatography to MS (LC-MS/MS), thus early biomedical work was limited due to the awkwardness of derivatization reactions, often following very lengthy sample preparation (extraction) steps. Robotic extraction based on 96-well technology has now become the standard in chromatographic methods of analysis, enabling the analysis of a few hundred samples per day per LC-MS/MS system. A review of the clinical chemistry literature and the symposia at clinical conferences today reveals an increase in the number of MS-related papers that cover clinical topics such as proteomics, drugs of abuse, and therapeutic drug monitoring.

The first established uses of MS in the clinical environment centered on determining concentrations of volatiles, including anesthetics. The detection of drugs of abuse in urine including athletic doping gained traction using GC-MS. Likewise, prior to 1980, screening for inborn errors of metabolism was performed on urinary extracts using GC-MS. GC-MS was the gold standard for newborn screening until the introduction of atmospheric pressure ionization methods (e.g., electrospray ionization [ESI]), which led to the coupling of liquid chromatography to mass spectrometry.1

Later, clinical screening for metabolic diseases in newborns became an established method based on LC-MS/MS and dried blood spots (DBS).2 To this day, DBS analysis continues and is receiving increasing interest from pharmaceutical companies as a method for determining the concentrations of xenobiotics in whole blood for early pharmacokinetics (PK) and toxicokinetics studies.3 While it is not an IVD, LC-MS/MS is the tool for assessing the PK of drugs in clinical trials and is used routinely for this purpose by contract research organizations and medical schools throughout the world.

More recently in fashion are direct atmospheric pressure desorption ionization MS methods.4 The principal technique is desorption electrospray ionization (DESI), which has been commercialized by Prosolia Inc. (Indianapolis; see Figure 1).5 Its uses span a wide range of applications from fingerprinting counterfeit tablets to detecting chemical warfare agents on surfaces and imaging thin sections of tissue for drugs and metabolites.6 A compelling aspect of this method is the ability to desorb molecules from virtually any type of surface, including steel, plastic, paper, or tissue. The DESI method offers many opportunities for the development of IVD tests for such applications as therapeutic drug monitoring in which blood is deposited onto absorbent paper and scanned in seconds using DESI-MS. While the technology is compelling and the applications are broad, both technical and economic challenges so far have limited its dissemination in the IVD industry.

Figure 1. An Omni Spray 2D DESI Ion Source coupled to a Thermo Scientific LTQ linear ion trap mass spectrometer.

Miniaturizing MS Instruments

Mass spectrometric measurements are performed on desolvated, gas-phase ions under vacuum conditions. In general, a mass spectrometer system consists of an ionization source for creating ions of the analytes in a sample, a mass analyzer for separating ions under the influence of electric or magnetic fields, and a detector that registers the number of ions. All of these components are housed in a vacuum system that is commonly evacuated to one billionth of atmospheric pressure or lower.

The mass analyzer is central, both figuratively and literally, to the mass spectrometer. A variety of choices are available on the commercial market, although there are trade-offs among them regarding size, cost, complexity, performance (stability, sensitivity, dynamic range, MS/MS capability, resolving power, and mass accuracy), and the skill required to use and maintain the instrumentation. For a bedside mass spectrometer, the ion trap may be well suited. Although some aspects of ion trap mass spectrometers’ (ITMS) performance may be disadvantaged when compared to high-performing analyzers, they offer some intrinsic advantages that make them a compelling candidate for miniaturized, clinically-based systems.

Generally, the performance of a mass spectrometer is related to the size of the mass analyzer. For example, time-of-flight mass spectrometers commonly improve mass resolution by increasing the length of the ion flight path. Conversely, reducing this length necessarily decreases the achievable mass resolution. Paul type ion traps of both the 3-D and more recently 2-D (linear) varieties offer users good mass spectral performance at a reasonable cost. In recent years, smaller field-portable MS units have become the focus of several research groups and mass-spectrometer vendors, with several finding their way to the commercial market. While nearly all of the mass analyzers have been miniaturized, most of the effort has focused on miniaturizing the ion traps.

Miniaturization of the ion trap is commonly achieved using simplified electrode geometries (e.g., the cylindrical ion trap), making the analyzer easier to manufacture while maintaining adequate performance for most applications. In addition, miniaturization of the ion trap results in the reduction of the operating voltage, allowing for smaller power supplies. Ion traps are typically operated at approximately 10-3 torr in a background of helium buffer gas. This operating pressure is at least two orders of magnitude higher than any other mass analyzer and is indeed necessary for getting the best performance from the ion trap analyzer.

The higher operating pressure reduces the vacuum pump requirements for miniaturized mass spectrometers with compact vacuum chambers, which results in smaller pumps than conventional mass spectrometers. This requirement is particularly important in miniaturized instruments featuring an atmospheric pressure interface for use with ionization sources such as ESI and DESI since the amount of gas to be pumped from atmospheric pressure to the operating vacuum level of the mass analyzer is significant. Lastly, ion traps are capable of performing MS/MS experiments in a single mass analyzer (i.e., tandem-in-time experiments), which eliminates the requirements for secondary mass analyzers and operating electronics that are needed for tandem in space experiments commonly performed with other mass analyzers.

As stated previously, smaller field portable MS units have recently come into favor and have made their way to the commercial market. Simplification of ion trap mass analyzers to cylindrical devices have lowered production costs, and with small instrument manifolds, larger roughing pumps are replaced with smaller drag pumps, which significantly reduces the size and weight of the overall package. These devices are now commercialized by instrumentation companies such as Griffin Analytical Technologies, a subsidiary of ICx Technologies (West Lafayette, IN), Torion Technologies Inc. (American Fork, UT), and Bruker Daltonics Inc. (Billerica, MA). However, mirroring the early quadrupole years, the applications are mainly focused on environmental and biodefense testing.

Mass Spectrometers for Point-of-Care Testing

What is envisioned is a point-of-care testing (POCT) system that includes a bedside mass spectrometer with automated blood sampling for critical care and therapeutic drug monitoring to optimize the dose of drugs with a narrow therapeutic index (e.g., chemotherapy, anticoagulants, and immunosuppressant anti-rejection drugs).

The analytes in a biological fluid can be divided into the following three types: conventional generic measures of patient status including electrolytes, oxygen, pH, glucose, etc.; disease-specific biomarkers that suggest a need for and a response to a particular intervention; and drugs and drug metabolites, including anesthetics. Detecting and measuring the chemical entities in the last two categories are where mass spectrometry demonstrates the most promise.

Therapeutic Drug Monitoring

Therapeutic drug monitoring (TDM) has been an important part of the IVD market since the late 1970s, but only for a few drugs for which the risks and costs justified the test. Most prescribed drugs are never monitored in patients, which is true for more than 99% of the patient population, even in the hospital setting. Thus, it is only a rough guess that the circulating concentration of a prescribed drug is adequate but not too high. Given that no two individuals are exactly alike and many phenotypes influence drug absorption, metabolism, and drug-drug interactions, it is a reasonable hypothesis that more TDM would be a very good thing if the technology and cost allowed for it. Mass spectrometry is the most promising in this area given that it is a physical method that does not rely on biological recognition, is applicable to the broadest range of analytes from small drugs to proteins, and has low limits of quantitation.

If the circulating concentration of a drug is determined in an individual subject versus time, the data captures all of the influences of genetics and disease on transporters, enzymes, etc., and can tightly link dose to an individual response. Getting the right concentration of a drug to be bioavailable in circulation is a concern. Even though TDM has been broadly available since the late 1970s, in most cases, the optimum response in an individual is still a guess based on the statistical average of a group in a trial, and time-dependent data is rarely examined in detail for individual patients.

For example, a determination of drug concentration may be made at a fixed point in time (e.g., 6 or 12 hours after dose), but not in any fashion that illustrates the dynamics. One caveat to keep in mind is that TDM covers all the influences of transporters, diet, fluid flows, and metabolism (including drug-drug interactions) on the drug in circulation, but it does not say anything about variations in the site of drug action from patient to patient. Thus, a lot of the factors that are very relevant to patient variation are covered, but not all of them.

A lot of attention has been given to the field of pharmacogenomics (personalized medicine, theranostics) to optimize drug selection and dose based on patient genetics. This includes obtaining a view of drug-metabolizing enzymes, including evaluating P450 isoforms via gene SNPs. The opportunities for improved therapy are great, and the pairing of therapy and IVDs will no doubt advance rapidly. But genes do not tell the whole story. For one thing, they interact in largely unknown ways, and protein expression is clearly affected by environmental and lifestyle considerations. Also, it makes little sense to do much genomic testing at the point of care. In the end, protein array chips may be more important than what can be learned from genes, but the technical problems are far greater to develop practical measurement devices. A pragmatic approach is still within reach, which does not require reductionist biology while an understanding of the mechanistic details evolves. This is technically, but not economically, feasible now.

The IVD industry is on the cusp of developing a critical-care plus TDM mass spectrometer that can optimize patient care in the next 5-10 years. The use of such technology in research, including drug trials, is currently possible if not affordable. TDM advanced rapidly in the 1980s based on enzyme immunoassay advances. But those tests have typically not been dynamic because there was no serial blood sampling, and the economics were unfavorable. That data was for a single point in time and was typically reported hours after the blood sample was drawn. This is still the case with very rare exceptions. Linking serial data to rapid decision making is becoming more feasible for critical care markers, disease markers, and drugs.

Clinicians generally want trend information regarding their patients’ blood pressure, temperature, pulse oximetry, and heart rate, which can be measured continuously or nearly so. When the same can be done for chemical parameters, it will be very powerful. The sensor community has long made this point, but there are fatal flaws with such an approach with respect to development, cost, quality assurance, concentration range, and limits to the number of analytes that can be tracked. POCT is highly developed today for the major analytes, such as pO2, pCO2, Na+, K+, Ca++, glucose, and lactate. Now it can be taken in new directions with more exotic and specific tests.

Technology is advancing very rapidly, and cost today is primarily limited by market size not technology. Mass spectrometry approaches and technologies are now becoming easier to use and less costly, and can deliver high sample throughput with minimal sample handling. New technologies such as DESI are potentially very promising (see Figure 1). DESI-MS enables rapid measurements for determining concentrations of xenobiotics in DBS and for determining drug and drug-metabolites’ spatial distribution in whole-body tissue sections from laboratory animals. Preliminary work for human cancer pathology samples toward intraoperative surgical guidance has also shown promising results. One example of the application of DESI-MS is the direct measurement of drugs of abuse in biological specimens. Figure 2 shows a DESI mass spectrum recorded on neonatal meconium after dissolution in methanol/water and drying onto a cellulose paper strip, confirming the presence of cocaine (m/z 304) in the sample.

Figure 2. A DESI mass spectrum of cocaine (m/z 304.1563; 500 ng/mL) present in neonatal meconium recorded using a Waters LCT Premiere benchtop mass spectrometer.

Conclusion

The feasibility of a bedside or near-patient mass spectrometer is real. It is real because the future of personalized medicine and disease treatment requires it. The broad adaptation of mass spectrometry is driving down instrument costs, while advancements in electronics, vacuum pumps, and ion motion control are enabling smaller, robust instruments to be developed. The convergence of novel turnkey sample introduction methods with purpose-built mass spectrometry-based systems will enable new measurements in IVDs, paving the way to personalized therapy and real-time therapeutic drug monitoring.

References

1. J Fenn, et al., “Electrospray Ionization-Principles and Practice,” Mass Spectrometry Reviews 9 (1990): 37-70.

2. DH Chase, TA Kalas, and EW Naylor, “Use of Tandem Mass Spectrometry for Multianalyte Screening of Dried Blood Specimens from Newborns,” Clinical Chemistry 49 (2003): 1797-1817.

3. B Barfield, et al., “Application of Dried Blood Spots Combined with HPLC-MS/MS for the Quantification of Acetaminophen in Toxicokinetic Studies,” Journal of Chromatography 870 (2008): 32-37.

4. RG Cooks, et al., “Ambient Mass Spectrometry,” Science 311 (2006): 1566-1570.

5. Z Takats, et al., “Mass Spectrometry Sampling Under Ambient Conditions with Desorption Electrospray Ionization,” Science 306 (2004): 471-473.

6. JM Wiseman, et al., “Desorption Electrospray Ionization Mass Spectrometry: Imaging Drugs and Metabolites in Tissues,” Proceedings of the National Academy of Sciences of the United States of America 105 (2008): 18120-18125.

Peter T. Kissinger, PhD, is chairman and chief executive officer at Prosolia Inc. (Indianapolis), and professor of chemistry at Purdue University. He can be reached at pkissinger@prosolia.com.
 

Brian C. Laughlin, PhD, is director of product development at Prosolia Inc. (Indianapolis). He can be reached at laughlin@prosolia.com.

Justin M. Wiseman, PhD, is director of research at Prosolia Inc. (Indianapolis) and co-inventor of DESI mass spectrometry. He can be reached at wiseman@prosolia.com.