Phillip R. Tiller

Accurate mass offers a significant advance over nominal-mass systems for metabolite studies. Accurate-mass data provide an increased ability to identify the metabolites observed through enhanced parent and fragment ion specificity. Examples provided show how accurate-mass data can differentiate between analytes of interest and the matrix. These examples also demonstrate how accurate-mass data of nonradiolabeled samples can address the challenges of in vivo metabolite identification studies previously only attempted with the combination of liquid chromatography–mass spectrometry and flow scintillation analysis. Further, a discussion of the use of accurate mass in glutathione profiling exemplifies the advantages of using accurate mass for in vitro applications.

In recent years, accurate mass–based analyses have become an increasingly important approach for qualitative mass spectrometry (MS) analyses (1–6). The use of accurate-mass applications has steadily replaced the use of nominal-mass approaches (7–11), and many scientists who are new to accurate-mass instrumentation are making greater use of the technologies now available. 

The goal of this paper is twofold. First, it describes the theoretical basis of why accurate-mass approaches offer a significant advance over nominal-mass approaches in the arena of qualitative analysis, and specifically metabolite identification. Second, it describes some of the analytical approaches that can now be conducted in a relatively routine manner — analytical approaches that were far from routine using the nominal-mass instrumentation. 

We should begin the discussion of accurate mass–based analyses with some key definitions, provided below. 					

 is the mass calculated using the average atomic mass of each element weighted for its natural isotope abundance.

 is the integer mass of the most naturally occurring stable isotope.

 is the calculated mass of an ion or molecule calculated using the mass of the most abundant naturally occurring isotope of each element.

is the calculated mass of an ion or molecule containing a single specified isotope of each atom.

 is the measured mass of an ion measured to high mass accuracy.

 is an MS technology that exhibits high mass resolving power.

The average mass is a value that is frequently used by medicinal chemists, but has no meaning in MS. No molecule exists that is represented by the average mass; the various isotopes present in a molecule  generate an isotope pattern in the mass spectrum. The terms 

 are often used interchangeably because the most abundant naturally occurring isotope commonly is used in calculating an exact mass. 

 describes the capability of a mass spectrometer to differentiate between adjacent masses such as 

 1000 and 1001. This term is appropriate when discussing the resolution of a quadrupole or ion-trap mass spectrometer.  

 that is appropriate for quadrupole orthogonal acceleration time-of-flight (QTOF) and orbital trap mass spectrometers is based on full width at half maximum (FWHM). Thus, 

 is defined as the mass being analyzed divided by the peak width of the raw mass data at that mass. So a mass spectrometer that detects an ion at 

 500 that has a peak width of 0.1 Da is operating at a resolving power of 500/0.1, or 5000. A mass spectrometer exhibiting a resolving power of 5000 can differentiate between 

Because mass resolution is a function of the mass of the ion being examined, when an instrument resolution is stated, the mass at which that resolution was determined also must be defined. In the example above, the mass spectrometer is exhibiting a resolution of 5000 at mass 500. 

Mass accuracy is a key parameter of mass spectrometer performance and determines the specificity of the mass measurement being made. The most commonly used definition of mass accuracy is given in parts per million (ppm) and is determined as the difference between an exact (or theoretical) and measured (accurate) mass. Table I displays the ppm errors for three different measured masses that have the same absolute mass error (25 mDa) relative to their exact masses. The ppm error, because it is a relative term, varies with the mass of the ion being examined. Traditionally, a mass error of 5 ppm has been accepted by medicinal chemistry journals as sufficient to provide a definitive elemental composition. Because 

 is a relative term, however, mDa mass accuracy, which is as an absolute measurement of instrument performance, often is used as a convenient day-to-day tool. 

 Table I: Example of ppm and mDa error at different masses 			

A mass spectrometer with high mass resolving power generally is considered an essential tool for achieving good mass accuracy. In reality, high resolving power is used to eliminate chemical interferences and is not otherwise required for obtaining good mass accuracy. In fact, accurate-mass measurements can be achieved routinely with even a quadrupole mass spectrometer operated at low mass resolving power if interferences are not a problem. However, given that the samples being analyzed usually are not pure standards but rather are mixtures or chromatographic peaks from LC–MS analyses, interferences are a frequent occurrence and HRMS is required to obtain quality accurate-mass data. 

The application of accurate-mass technology offers several advantages in conducting metabolite identification studies when compared with the use of nominal-mass instrumentation such as triple-quadrupole or ion-trap mass spectrometers.   

A review of the three main goals in conducting metabolite identification studies creates a framework for understanding these advantages. These goals are the same whether the samples are derived from in vitro or in vivo experiments. 

The first goal of metabolite identification is to differentiate between ions derived from the drug itself and drug-related metabolites and ions derived from the matrix. The second goal is to identify the biotransformation that has taken place, such as addition of an oxygen atom or loss of a methyl group. The third goal is to define which site of the drug molecule has undergone biotransformation. Accurate-mass data enable all three of these goals to be achieved more quickly than when using nominal-mass data. 

A paradigm shift is possible in using accurate-mass approaches because the data are rarely compromised with interferences and thus provide more information about the molecule being analyzed. Compared to nominal-mass studies, the overall result is that conclusions are reached more rapidly, with a higher level of confidence in their accuracy. 

There are four main reasons that accurate-mass data are superior to nominal-mass data in conducting metabolite identification studies, and these are discussed below. 

How is it that having accurate-mass data enables differentiation between the analytes of interest and the matrix? In most instances, the matrix components to be eliminated as false positives are derived from biological sources such as microsomes, hepatocytes, plasma, or urine. Such components are predominantly made of carbon, hydrogen, nitrogen, and oxygen. 

Table II: List of common elements and their accurate masses 			

Most xenobiotics that are being analyzed, though mainly derived of the same elements, often contain a higher level of unsaturation and occasionally incorporate halogens. Table II lists some basic parameters of common elements. Carbon does not contribute to the fractional mass (the numerals after the decimal point) of a molecule's mass, whereas both hydrogen and nitrogen increase the fractional mass, and oxygen, halogens, and sulfur decrease the fractional mass. Thus, the fractional mass of a molecule will change with the elements that constitute the structure of the compound. 

For example, for each double bond introduced into a molecule, the fractional mass will decrease by 0.0156 Da. Because many of the xenobiotics that are drug candidates are more highly aromatic than naturally occurring "interferences" such as peptides, the observed fractional mass is lower. A stylized example of this is shown in Figure 1, in which a tripeptide, Tyr-Gly-Gly, is compared with a possible drug compound of similar nominal mass. The mass difference of 32 mDa would be easily differentiated using a modern accurate-mass instrument. 

Figure 1: Example of a tripeptide and a possible xenobiotic with similar nominal mass. 			

The difference in fractional mass enables elimination of false positive mass "hits" when dealing with biological matrices. Figure 2 reproduces two extracted ion chromatograms from the same data file. The top trace uses a mass window of ±0.5 Da and the bottom trace uses a mass window of ±0.005 Da. The nominal-mass top trace has multiple peaks or "hits" for the parent compound, whereas the accurate-mass bottom trace has only one peak. 

Figure 2: Two extracted ion chromatograms from the same data file. The top trace is generated using a 1-Da wide window. The bottom trace is from using a 10-mDa wide window.			

Accurate-mass data facilitate differentiation of biotransformations that result in an identical nominal-mass shift. For example, when using a nominal-mass instrument, it is impossible to differentiate between M + O – 2H or M + CH

 because the mass shift (in both instances) is +14 Da. When the same comparison is made using accurate-mass data, the difference is very clear, because M + O – 2H (M + 13.9793) or M + CH

 (M + 14.0156) results in a mass shift that differs by 36 mDa. 

An essential step in conducting a metabolite identification study is to determine which fragment ion in the MS-MS spectrum is derived from the correct substructural moiety of the parent molecule. This information is used to compare the MS-MS spectra of a metabolite with the parent drug and thus determine the part of the molecule that has undergone biotransformation. The use of accurate-mass data in this step can prevent errors in localization of the biotransformation, because nominal-mass data cannot differentiate between different substructures in the molecule that provide the same nominal-mass fragment ions. 

Figure 3 shows a molecule that would provide fragment ions with similar nominal mass that would require an accurate-mass analysis to be correctly interpreted. Incorrectly interpreting the MS-MS spectrum of the parent drug would result in incorrect localization of the biotransformation. An incorrect localization of the site of metabolism could lead to synthetic chemisty decisions to address a metabolic instability that would alter the wrong site on the molecule. 

Figure 3: Example of a potential drug molecule that provides multiple fragment ions with the same nominal but different exact masses. 			

Nominal-mass instruments use MS-MS methods to obtain specificity, such as multiple reaction monitoring (MRM) for quantitative assays and precursor ion scans and constant neutral loss scans for qualitative screening or identification. These MS-MS approaches are less sensitive than full-scan experiments, simply as a consequence of transmission efficiency in such MS-MS instrument modes. The application of accurate mass using full-scan experiments is more sensitive than nominal-mass MS-MS methods because equal, or superior,  specificity is obtained without the resultant loss of ion current. 

The Role of Metabolite Identification in Various Stages of Drug Development

Metabolite identification plays an important role throughout the discovery, preclinical, and clinical stages of small-molecule drug development (see Figure 4). 

Figure 4: Schematic of different metabolite identification experiments performed throughout the drug discovery, preclinical, and clinical stages of drug development. 			

In drug discovery, metabolite identification can be used to determine the routes of excretion of a drug and its metabolites, to define the sites on a molecule that are subject to metabolism, to assess the potential for formation of reactive metabolites, and to compare the metabolites formed across various species, including humans. 

In preclinical drug development, in vivo studies, typically in rats, dogs, and monkeys, are designed to confirm that the metabolite profile observed across species in the in vitro studies are also observed in vivo. The mass balance study also illuminates the routes of excretion and their relative quantitative importance. 

The clinical development role of metabolite identification is generally twofold. First, it determines that the circulating metabolites observed in humans were also present in the toxicity studies filed in the investigational new drug (IND) application, thus demonstrating that the toxicity of the human metabolites has been tested. Second, it defines the structure of the metabolites observed in human plasma, urine, and feces and the relative importance of the observed route of excretion. 

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Accurate-mass approaches offer a significant advance over nominal-mass approaches in the arena of qualitative analysis, and some of the analytical approaches can now be conducted in a relatively routine manner.

Phillip R. Tiller

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Accurate Mass: Why It's the Best Solution for Metabolite Identification in Discovery, Development, and Clinical Applications