Achieve Accurate Quantification in Mass Spectrometry | Techniques & Methods

In quantitative analysis, it is essential to establish a correlation between the measured signal intensity and the concentration of the analyte. Mass spectrometry (MS) facilitates this correlation, allowing signal intensity to be utilized for quantitative analysis.

Mechanism of Quantification in Mass Spectrometry

The quantification process in mass spectrometry can be categorized into three distinct phases: ion generation, ion transmission, and ion detection.

Ion Generation

During ion generation, the processes by which different sample molecules are converted into ions occur independently of one another. Consequently, an increase in sample concentration results in a proportional increase in the number of generated ions. Various ionization techniques currently employed, such as Electron Ionization (EI), Inductively Coupled Plasma (ICP), Electrospray Ionization (ESI), and Matrix-Assisted Laser Desorption/Ionization (MALDI), demonstrate a linear relationship between sample concentration and ion yield, applicable within specific concentration ranges.

Ion Transmission

The transmission of ions is characterized by an efficiency that is largely independent of the quantity of ions being transmitted. However, it is noteworthy that excessive ion transmission may lead to mutual repulsion among similarly charged ions, resulting in an expansion of the ion beam and a subsequent decrease in transmission efficiency. This phenomenon is particularly pronounced in confined ion traps, necessitating the implementation of critical techniques to regulate the number of ions introduced into the mass spectrometer, ensuring that the ion quantity remains within optimal limits.

Ion Detection

In the ion detection phase, irrespective of whether a photomultiplier tube or an Ion Cyclotron Resonance (ICR)/Orbitrap detector is utilized, there exists a linear correlation between signal intensity and ion quantity, provided that the quantities remain within a defined range.

Clarification on Relative and Absolute Signal Intensity

A common misconception regarding the quantitative capabilities of mass spectrometry arises from the representation of mass spectra using relative intensity, typically ranging from 0 to 100%, with the strongest peak normalized. However, it is crucial to note that the mass spectrometer records absolute intensity, from which relative intensity is derived. For quantitative analysis utilizing absolute intensity, attention must be directed toward this often-overlooked aspect of the data.

Coupling of Chromatography and Mass Spectrometry

In the context of chromatographic techniques coupled with mass spectrometry, the relationship between the analyte and the measured signal acquires an additional layer of complexity. The process can be articulated as follows: analyte concentration → concentration of sample in chromatographic effluent → mass spectrometry signal. Unlike the primarily relative intensity analysis of EI mass spectra, the absolute intensity of signals becomes critical in chromatography-mass spectrometry (GC-MS or LC-MS) applications. The temporal variation of mass spectrometry signal intensity is represented as a chromatogram, commonly visualized as total ion current or the intensity of specific mass-to-charge ratio (m/z) ions.

Total Ion Current (TIC) representation

The figure referenced is known as the Total Ion Current (TIC) representation. In this graphical format, the abscissa denotes time, while the ordinate represents signal intensity. It is pertinent to recognize that mass spectrometry data encompasses three dimensions: time, signal intensity, and mass-to-charge ratio (m/z).

In the process of peak detection, it is essential to select a specific m/z range from the TIC. This specialized selection is referred to as the Extracted Ion Chromatogram (EIC). The EIC facilitates the analysis of ions corresponding to particular m/z values over the designated time interval, thereby enhancing the clarity and specificity of the data interpretation.

This delineation of TIC and EIC is fundamental in mass spectrometric analyses, enabling the identification and quantification of analytes with increased precision.

Mass trace and chromatographic peak of analyte of interest

The analyte of interest exhibits a mass-to-charge ratio (m/z) within the range of 285.065 to 285.080. The upper figure illustrates the correlation between m/z values and retention time, while the lower figure depicts the relationship between signal intensity and retention time. Each point represented in the figures corresponds to an ion detected by the mass spectrometer.

The formation of a peak, as shown in the lower figure, indicates the presence of a substance at that specific retention time. The signal intensity of a particular analyte is typically quantified by the area under the peak. Alternatively, signal intensity may also be represented by the highest intensity point, denoted in red.

This approach to interpreting mass spectrometric data underscores the utility of peak area and maximum intensity as metrics for analyte quantification, facilitating accurate analytical assessments.

Quantitative Methods in Mass Spectrometry

The ability of MS to perform quantitative analysis has been established. This section will delineate the specific quantitative methodologies employed in mass spectrometry. The quantification process of any physical quantity fundamentally involves comparison against a standard, and mass spectrometry is no exception. Two principal methods are typically utilized for quantitative analysis: the external standard method and the internal standard method.

External Standard Method

The external standard method involves conducting experiments with a known quantity of standard sample A and an unknown quantity of analyte A. This approach yields three critical pieces of information: the amount of the standard sample (known), the signal intensity of the standard sample, and the signal intensity of the unknown sample. Assuming a linear response where the sample response is proportional to concentration, these three parameters can be utilized to calculate the quantity of the unknown sample.

To enhance the precision of the determination of the unknown sample, it is desirable for the signal intensity of the standard sample to closely approximate that of the unknown sample. This alignment minimizes the effects of non-linear responses. Therefore, in practice, the external standard method often entails measuring a series of known standard samples, from which a calibration curve is constructed. Subsequently, this curve is employed to estimate the quantity of the unknown sample through fitting techniques.

An example of a standard curve showing the absorbance of different concentrations of protein (two trials for each measurement). (WIKI)

Internal Standard Method

The external standard method is subject to two primary limitations:

1. The standard sample and the unknown sample are analyzed independently, making it impossible to eliminate random errors that may arise between experiments.
2. The matrix, defined as the components other than the analyte of interest, may differ between the standard and the unknown sample, potentially introducing additional discrepancies.

To address these limitations, the internal standard method is employed. In this method, a known quantity of standard sample B is directly introduced into the unknown sample A, allowing both the standard and the analyte to be analyzed within the same experiment and under identical matrix conditions. This approach effectively eliminates errors arising from differences in experimental conditions and matrix effects.

In cases where the standard sample and the unknown analyte are indistinguishable, quantification cannot be achieved through a single analysis. Instead, measurements must be taken both before and after the addition of the internal standard. This involves recording the signal intensities of the unknown sample alone and of the mixture containing both the unknown and the internal standard. Such measurements facilitate the calculation of the unknown sample quantity with increased accuracy.

In conclusion, both the external and internal standard methods provide valuable frameworks for quantitative analysis in mass spectrometry, each with distinct advantages and limitations that can be strategically leveraged depending on the analytical requirements.

Quantitative Details Related to Mass Spectrometry

Isotope Dilution

In the previous discussion regarding the internal standard method, it was noted that the ideal internal standard must be both similar to and distinct from the analyte of interest; this contradictory requirement is fulfilled by the use of isotopic internal standards.

Compounds composed of different isotopes exhibit nearly identical physicochemical properties, and their ionization responses are typically comparable. However, they possess distinct mass-to-charge ratios (m/z), which allows for differentiation within the mass spectrometer. Thus, isotopic standards serve as the most suitable internal standards.

Furthermore, due to the natural abundance distribution of certain elements' isotopes, the introduction of a known quantity of isotopic internal standard allows for the conversion of absolute intensity measurements into relative proportion measurements. This conversion enhances the accuracy of the analytical results.

Basic principle of isotope dilution

Selected Reaction Monitoring

In relatively simple systems, qualitative identification of a compound may be achieved based solely on its molecular weight. However, in complex mixtures, such as petroleum products or biological samples, many compounds share identical or nearly identical molecular weights. Isomers can exhibit the same mass, and compounds such as carbon monoxide (CO) and nitrogen (N₂) may require consideration of the instrument's mass resolution to distinguish between them. Therefore, measuring mass alone is insufficient to confirm whether a compound is the target analyte.

In tandem mass spectrometry (MS/MS), the mass spectrometer functions not only as a balance for weighing ions but also as a tool equipped with "tweezers" for selectively isolating specific ions and "scissors" for activating and fragmenting particular ions.

Through the sequential selection of parent ions and daughter ions, precise identification of the target compound can be achieved within complex matrices. This dual ion selection also mitigates interference from the complex matrix and reduces background noise, thereby enhancing detection limits and improving the dynamic range of the method. Consequently, selected reaction monitoring (SRM) is currently one of the most widely employed quantitative methodologies in GC-MS or LC-MS applications.

Schematic representation of tandem-MS/MS

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