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Mass Data Terminology, Considerations, and Interpretation

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Mass Data Terminology, Considerations, and Interpretation MASS DATA TERMINOLOGY, CONSIDERATIONS, AND INTERPRETATION Monolinuron structure Terminology Definition Monoisotopic The sum of the lowest isotopic mass of the atoms in a molecule. For most small molecules, the monoisotopic ion mass is the most abundant (base peak) in the spectrum. For larger molecules, such as peptides, the base peak ion may be different. Monolinuron (monoisotopic mass) = 214.05 Da. Average mass The mass obtained by summing the constituent atoms in a molecule, using their average atomic mass. The Monolinuron average atomic mass can differ significantly from the monoisotopic mass. For example, the average mass of 35 37 (C₉H₁₁ClN₂O₂) is used as chlorine is based upon the relative abundance of its two naturally occuring isotopes, Cl and Cl, (34.97 x 24%) an example in this + (36.97 x 76%) = 35.45 Da. Since the average mass is a calculated value, a corresponding peak does not appear document to in the spectrum (see Figure 1). Monolinuron (average mass) = 214.65. demonstrate some Nominal mass The monoisotopic mass rounded up or down to the nearest integer. Monolinuron (nominal mass) = 214 Da. principles of MS spectrum + Mass to charge The QDa® mass scale is in m/z and not Daltons. For the singly-charged monoisotopic ion [M+H] of monolinuron, interpretation. ratio (m/z) m = 214.05 and z = 1. Therefore, a peak will appear on the spectrum at [214.05+1.0078]/1 = 215.06. [M+H]+ ion The positively charged form of a molecule that is created when one proton is added. The ion detected is one unit higher than the monoisotopic mass of the uncharged molecule. [M-H]- ion The negatively charged form of a molecule that is created when one proton is removed. The ion detected is one unit lower than the monoisotopic mass of the uncharged molecule. Dalton The unit of atomic mass. Formula weight The listed formula weight can include contributions of hydrates and salts. In such cases, an ion associated with considerations formula weight does not appear in the spectrum. For example, the empirical formula of clopidogrel hydrogensulfate is C16H16ClNO2S•H2S04 and the formula weight is listed as 419.90. However, because of dissociation of salt in the solution, the mass spectrum presents an [M+H]+ ion at m/z 322, which corresponds to + [C16H16ClNO2S+H] . Table 1: Monolinuron average mass and monoisotopic mass comparisons Figure 1: Differences between the average and monoisotopic mass of monolinuron Element Isotopic masses Atomic mass Atomic mass (% Abundance) (Average) (Monoisotopic) C 12.0000 (98.90%) 12.0110 12.0000 [M+H]+ = 215.06 (monoisotopic) 13.0034 (1.10%) H 1.0078 (99.99%) 1.0079 1.0078 [M+H]+ = 215.66 (average) 2.0141 (0.015%) CI 34.9689 (75.77%) 35.4527 34.9689 37 36.9659 (24.23%) Peak due to CI isotope monolinuron N 14.0031 (99.63%) 14.0067 14.0031 mass 15.0001 (0.37%) O 15.9949 (99.76%) 15.9994 15.9949 For most small molecules, the base peak corresponds to the monoisotopic 16.9991 (0.038%) ion ([M+H]+ or [M-H]-). The average mass does not actually appear in the 17.9992 (0.20%) spectrum. For larger molecules, such as peptides, the monoisotopic mass Sum (M or neutral C₉H₁₁ClN₂O₂ 214.65 214.05 may not be the base peak. molecule) Sum [M+H]+ C₉H₁₁ClN₂O₂ + [H]⁺ 215.66 215.06 Continuum and centroid data In-source fragmentation Continuum data shows a full profile of the distribution of ions present Peaks observed at a lower m/z value to the analyte can correspond to fragments in a spectrum. Centroid data (or stick data) is continuum data processed of the analyte generated in the source. These fragment ions are characteristic of to display a single, centered point for each distribution of ions in a the analyte. Higher proportions of fragment ions are observed at higher cone spectrum. voltages as more energetic collisions occur in the source. Figure 2: Continuum and centroid data overlaid Figure 3: In-source fragmentation Monolinuron [M+H]+ Monolinuron Monolinuron fragment ion [M+H]+ Centroid and continuum -89 spectra overlaid mass mass Dimers Multiple charges Spectral peaks can be observed at almost double the expected The QDa mass scale is m/z and not Daltons. For singly-charged species (z = 1), the monoisotopic ion m/z value. These dimers are formed by the association observed mass corresponds to the monoisotopic mass. For larger molecules, it is of an ion such as [M+H]+ with its neutral counterpart, for example, common to see higher charge states, such as z = 2 or z = 3. Therefore, a series of M+[M+H]+. Dimers are often only formed at high concentrations. If progressively smaller m/z peaks can be observed, corresponding to the same observed, consider diluting the sample or injecting less. analyte with different charge states. Figure 4: Dimers Figure 5: Multiple charging with angiotensin II peptide [M+H]+ Singly charged ion. Monolinuron Monolinuron dimer 2+ + [M+2H] Note the 1 m/z isotope [M+H] M+[M+H]+ Doubly charged spacing. ion. Isotope spacing is 0.5 m/z N charges: m/z = [M+nH]/n and isotopic resolution is not achieved. mass mass Waters, The Science of What’s Possible, and QDa are registered trademarks of Waters Corporation. All other trademarks are the sole property of their respective owners. ©2015 Waters Corporation. Produced in the U.K. August 2015 715004945 Rev. A MASS DATA TERMINOLOGY, CONSIDERATIONS, AND INTERPRETATION Adduct ions Table 2: Common adducts and where they exist in a spectrum These ions are formed by the association of metal ions, or other species Common positive ion m/z observed m/z difference from contained in the eluent, with analytes in the ESI source. The formation and adducts [M+H]+ abundance of the adduct depends on the chemistry of the analyte and mobile + phase components. Adduct formation can increase with lower quality solvents. [M+H] M+1 0 + Figure 6: Adduct ions [M+NH4] M+18 +17 [M+Na]+ M+23 +22 [M+K]+ M+39 +38 Monolinuron [M+H]+ Common negative ion m/z observed m/z difference from +22 - adducts [M-H] [M-H]- M-1 0 Monolinuron [M+CI]- M+35 +36 [M+Na]+ [M+HCOO]- (formate) M+45 +46 mass - [M+CH3COO] (acetate) M+59 +60 Terminology Definition Capillary voltage The voltage applied to the electrospray capillary to promote the ionization process. Cone voltage A focussing voltage applied to the ion guide, transferring ions generated in the source into the QDa. Probe temperature The temperature applied to the electrospray probe to promote mobile phase evaporation as part of the ionization process. MS scan (or full scan) Records all of the masses within a user-defined range at each time point in the chromatogram to produce qualitative information. An MS scan can be performed in positive (where only positively charged ions are transmitted and detected) or negative ion mode. This is similar to 3D PDA data. Single ion recording (SIR) Records a single user-defined m/z value (positive or negative ion mode) at each time point in the chromatogram, with all other ions being rejected. This technique can introduce specificity and higher sensitivity into an analysis, and is most commonly used for quantitative analysis where lower limits of detection are required. This is similar to 2D PDA data. Extracted ion chromatogram (XIC) A chromatogram, extracted from an MS scan, that only displays the intensity of a selected mass. Total ion chromatogram (TIC) A chromatogram in which the intensity is displayed as a sum of all individual ion intensities. Sampling rate or sampling The number of data points per second acquired for each MS experiment function, such as SIR or MS scan, that you specify. frequency (Hz) Mass scale calibration The mass scale is calibrated against a series of known masses. The calibration is required to ensure that the theoretical and observed masses correspond. You can automatically configure a mass scale calibration check upon instrument start-up. Source saturation Source saturation occurs as a result of the ionization efficiency changing at high concentrations. Source saturation is a gradual effect where a reducing proportion of molecules are ionized as the concentration increases, which results in non-linearity. Figure 7: Source saturation Circled calibration points showing source saturation. Response Conc Detector saturation Detector saturation occurs when the signal arriving at the detector exceeds an upper, finite limit. When you display spectral data in continuum mode, flat- topped peaks appear. In centroid mode, you can indentify detector saturation when the intensity of an isotopic peak such as 13C, relative to the monoisotopic peak, is higher than theoretically predicted. Figures 8 and 9 display unsaturated and saturated spectral peaks from sulfadimethoxine injections. Figure 8: An unsaturated spectral peak from a sulfadimethoxine Figure 9: A saturated spectral peak from a sulfadimethoxine injection injection (showing continuum data inset) The apex of the peak is missing; therefore, the Unsaturated peak annotated m/z value may not be accurate. Observed relative The saturated intensity of 13C peak peak displaying a is higher than the flat-top profile. theoretical percentage. m/z m/z .
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