Quantitative Measurements by Mass Spectrometry

In a large portion of routine and discovery-oriented analyses, mass spectrometry (MS) is used as a qualitative technique. The obtained qualitative data enable detection and structural elucidation of molecules present in the analyzed samples. However, modern chemistry and biochemistry heavily rely on quantitative information. In biochemistry it is often sufficient to conduct quantification of analytes in biofluids every few hours, days, or even weeks. In the real-time monitoring of highly dynamic samples, it is necessary to collect data points at higher frequencies. When it comes to selection of techniques for quantitative analyses, especially in the monitoring of dynamic samples, MS has not generally been favored. In fact, the performance of MS in quantitative analysis is worse than that of optical spectroscopies - especially, ultraviolet-visible (UV-Vis) absorption and fluorescence spectroscopy. 

The limitations of MS in the acquisition of quantitative data (absolute or relative) are due to several factors which can be categorized into two groups encompassing those related to the instrument (I) and the sample (II). The contributions of different factors are outlined below. 

Time-Resolved Mass Spectrometry From Concept to Applications, First Edition. Pawel Lukasz Urban, Yu-Chie Chen and Yi-Sheng Wang. 

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The gas-phase concentrations of reactants (NO, NO2 and N2O) and products were measured by mass spectrometry. Mass numbers 30 of NO, 46 of NO2, 44 of N2O, 28 of N2 and 32 of O2 were used for the quantitative analysis. It is important to emphasize that no detectable Oz was found in NO decomposition at the reaction conditions used. 

A number of methods are used in classical analysis to perform these tasks. Qualitative as well as quantitative analysis of mixtures can be achieved by chromatographic methods such as gas chromatography (GC) and liquid chromatography (LC). Chemical sensors or biosensors can also be employed for selectively quantifying a compound in a mixture. However, such analysers have only been developed for a very limited number of analytes. Identification of pure compounds can be achieved by nuclear magnetic resonance (NMR) measurements, by mass spectrometry (MS), infrared spectroscopy (IR), UV/vis spectroscopy or X-ray crystallography, to name a few. 

Boyd RK, Basie C, Bethem RA. Measurement, dimensions and units. In trace quantitative analysis by mass spectrometry. New Jersey Wiley 2008. pp. 6-8. 

Every ionization method exhibits compound-dependent ionization efficiencies (Chap. 2.4). Whether a specific compound is rather preferred or suppressed relative to another greatly depends on the ionization process employed to deliver the ions to the mass analyzer. These circumstances require a careful calibration of the instrument s response versus the sample concentration for correct quantitation [6,7,50]. While relative signal intensities are perfect for qualitative analysis, i.e., for compound characterization, some means of measuring absolute intensities would be preferred in quantitation. Basic considerations on how to approach a quantitative analysis by mass spectrometry are given below [51-54]. Readers interested in a treatment of all aspects of quantitative analysis by mass spectrometry may refer to the highly recommended book by Boyd, Basic, and Bethem [50]. 

This technique provides quantitative information about tautomeric equilibria in the gas phase. The results are often complementary to those obtained by mass spectrometry (Section VII,E). In principle, gas-phase proton affinities, as determined by ICR, should provide quantitative data on tautomeric equilibria. The problem is the need to correct the measured values for the model compounds, generally methyl derivatives, by the so-called N-, 0-, or S-methylation effect. Since the difference in stability between tautomers is generally not too large (otherwise determination of the most stable tautomer is trivial) and since the methylation effects are difficult to calculate, the result is that proton affinity measurements allow only semi-quantitative estimates of individual tautomer stabilities. This is a problem similar to but more severe than that encountered in the method using solution basicities (76AHCS1, p. 20). 

Only arc/spark, plasma emission, plasma mass spectrometry and X-ray emission spectrometry are suitable techniques for qualitative analysis as in each case the relevant spectral ranges can be scanned and studied simply and quickly. Quantitative methods based on the emission of electromagnetic radiation rely on the direct proportionality between emitted intensity and the concentration of the analyte. The exact nature of the relation is complex and varies with the technique it will be discussed more fully in the appropriate sections. Quantitative measurements by atomic absorption spectrometry depend upon a relation which closely resembles the Beer-Lambert law relating to molecular absorption in solution (p. 357 etal.). 

Elemental composition C 52.96%, 0 47.04%. It may be analyzed by treatment with water. The product malonic acid formed may be measured quantitatively by direct injection of aqueous solution into a GC for FID detection. Alternatively, the aqueous solution may be evaporated and the residue may be derivatized to methyl ester and identified by mass spectrometry. Also, the gas may react with ammonia or an amine, and the amide derivative may be identified and quantitatively determined by GC-FID, GC-NPD, GC/MS or infrared techniques. 

An unknown substance, X, was isolated from rabbit muscle. Its structure was determined from the following observations and experiments. Qualitative analysis showed that X was composed entirely of C, H, and 0. A weighed sample of X was completely oxidized, and the H20 and C02 produced were measured this quantitative analysis revealed that X contained 40.00% C, 6.71% H, and 53.29% O by weight. The molecular mass of X, determined by mass spectrometry, was 90.00 u (atomic mass units see Box 1-1). Infrared spectroscopy showed that X contained one double bond. X dissolved readily in water to give an acidic solution the solution demonstrated optical activity when tested in a polarimeter. 

SFC provides complementary quantitative data to the structural information afforded by mass spectrometry. Thermally label materials such as isocyanates can be easily analyzed with minimal sample preparation. Supercritical carbon dioxide is nontoxic and can be obtained in high purity as measured by FID. The easy coupling of SFE with SFC makes the selective isolation and quantification of targeted analytes possible. Furthermore, we are in an age of increased environmental awareness. Solvent disposal is discouraged and has become very expensive. The waste disposal costs associated with supercritical carbon dioxide are negligible when compared to the solvent disposal costs generated by traditional Soxhlet methods. 

Unknown organic compounds can be identified and quantitatively measured by gas chromatography/mass spectrometry (GC/MS). Analytes can be thermally desorbed from the adsorbent surface and analyzed by GC/MS. Alternatively, the bulk air from the site collected in a Tedlar or a canister can be injected or introduced by heating or suction into the system for separation and analysis. 

Albumin can be measured quantitatively by the bromcresol green method in most species (Evans and Duncan 2003). Other proteins (described below) are measured by immunometric methods. Newer methods based on proteomics technology (concentration of proteins by acetone precipitation or ultracentrifugation, separation by 2-d gel electrophoresis or chromatographic techniques with subsequent identification and quantitation by mass spectrometry) have been used experimentally (Bandara and Kennedy 2002 Chapman 2002 Thongboonkerd et al. 2002a, b). 

Quantitative analysis has become possible due to technical advances in synthesis of complex molecules with isotopic labels at any one of many specific position and measurements of KIE determined accurately and precisely by mass-spectrometry and radioactive methods. The most informative method for elucidation of the enzyme reaction limiting step and nature of transition-state is the competitive labeled method (Schramm, 1999). This method is based on the use of two labeled preparations of the same substrate, one with the labeled atom at a site expected to experience bonding changes at the TS and a second preparation with a different labeled atom at a site remote from the bond-breaking site. Many molecules of interest can be specifically labeled with radioactive atoms T or I4C and can be incorporated into substrates that also contain stable isotopes D, 15N and 180. 

Determination of free 4-hydroxy-2,3-trans-alkenals by HPLC Esterbauer (1982) has developed a procedure for the qualitative detection and quantitative measurement of steady-state concentrations of free hydroxyalkenals (specifically HNE) in tissues, tissue extracts and lipid containing foodstuffs. Their method utilizes UV-detection of the free aldehyde at its 220 nm UV-absorption maximum and peak identification was confirmed by mass spectrometry. An effective purification and concentration step is employed using dichloromethane to extract hydroxyalkenals from samples trapped on Extrelut columns. The samples are subsequently purified by solid-phase extraction on octadecyl-bonded silica (ODS) disposable cartridges and then analysed by HPLC. 

Steen H, Jebanathirajah JA, Rush J, Morrice N, Kirschner MW. Phosphorylation analysis by mass spectrometry Myths, facts, and the consequences for qualitative and quantitative measurements. Mol. Cell. Proteomics 2006 5 172-181. 

We have also determined the upper limit of deuterium incorporation in HCA II. For this purpose milligram quantities of H labeled protein were produced in defined media containing 98.8% D2O and [ Hs, 98%] sodium acetate as the sole carbon source using the optimized procedures outlined above. To quantitate the level of deuterium incorporation, we analyzed the molecular mass of purified HCA II by mass spectrometry. The molecular mass of fully protonated HCA II was measured to be 29102 +/- 2.4 (theoretical mass = 29098.9). At low pH the protein contains 2018 protons therefore, one would predict a theoretical mass increase of 2030.5 mass units upon complete deuteration. The molecular mass of protein produced in 98.8% D2O and ["H3, 98%] sodium acetate was measured to be 31133 +1-13, an increase of 2034 +/- 15 mass units, indicating above 96% deuterium incorporation. 

Cellular Metabolites. - A review of methods for the measurement of ml has been produced with 95 references. It examines the quantitative measurement of ml by mass spectrometry and in vivo NMR. The NMR chemical shifts and /-coupling values of 35 metabolites which can be detected by in vivo or in vitro investigations of the mammalian brain have been published. The principles and recent applications of dynamic nuclear polarisation, which combines the sensitivity to oxygen of EPR and the tractability of NMR imaging, have been reviewed with 244 references. A review of studies of intermediary metabolism, including the use of NMR in the analysis of substrate selection under in vivo conditions, has been produced. A review has been produced, with 74 references, on the study of metabolic flux and subcellular transport of metabolites using NMR. " ... 

The gas analysis system consisted of an FT-IR spectrometer (Bruker IFS-66) with a heatable gas cell (100 cm volume) and a quadrupole mass spectrometer (Balzers GAM 400). NO, NO2, N2O, NH3, CH4, C3H6, C3H8, CO, CO2, and H2O were analysed by FT-IR spectroscopy and O2 and H2 by mass spectrometry. The analytical system permitted the quantitative analysis with a resolution of up to 15 measurements per second. 

Owing in part to the relatively recent development of cluster chemistry, there is an almost complete lack of thermodynamic data for cluster compounds. The measurement of appearance potentials of positively charged ions by mass spectrometry has offered a technique for ready measurement of this parameter, but the high values obtained for ionization potentials and heats of formation of ions by this method cast some doubt on its accuracy (358). It would appear that ions are generated in excited states (22). It may be expected that there will be a considerable increase in the thermodynamic investigations of clusters in the near future, so as to provide quantitative data on which to base theoretical considerations. 

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