Mass internal calibration

Elemental compositions are preferably assigned via internal mass calibration. The calibration compound can be introduced from a second inlet system or be mixed with the analyte prior to analysis. Mixing calibration confounds with the analyte requires some operational skills in order for it not to modify the analyte or to be modified itself. Therefore, a separate inlet for introducing the calibration compound is preferred. This can be done by introducing volatile standards such as PFK from a reference inlet system in electron ionization, by use of a dual-target probe in fast atom bombardment, or by use of a second sprayer in electrospray ionization. 

Note Mass accuracy may suffer from too high settings of resolving power if this causes noisy peaks. Often centroids are determined more accurately from smooth and symmetrically shaped peaks at moderate HR. One should be aware of the fact that the position of a peak of 0.1 u width, for example, has to be determined to V50 of its width to obtain 0.002 u accuracy [32]. 

Basic Rules The assignment of molecular formulas from accurate mass 

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Internal mass calibration typically yields mass accuracies as high as 0.1-0.5 mmu with Fourier transform ion cyclotron resonance, 0.5-5 mmu with magnetic sector, and 2-10 mmu with time-of-flight analyzers. 

Fig. 6.34. LR- (a) and HR-EI (b) mass spectra of 2-(l-methylpropyl)-phenol. The elemental compositions as obtained from accurate mass measurement are directly attached to the corresponding peaks. Peaks with small-lettered labels belong to PFK and residual air used for internal mass calibration (Chap. 3.3).

Fig. 9.13. Positive-ion FAB spectrum of a cationic fluorescent marker dye with PEG 600 admixed for internal mass calibration. By courtesy of K. H. Drexhage, University of Siegen and J. Wolfrum, University of Heidelberg.

MALDI is the method of choice for the analysis of synthetic polymers because it usually provides solely intact and singly charged [62] quasimolecular ions over an essentially unlimited mass range. [22,23] While polar polymers such as poly(methylmethacrylate) (PMMA), [83,120] polyethylene glycol (PEG), [120,121] and others [79,122,123] readily form [M+H] or [M+alkali] ions, nonpolar polymers like polystyrene (PS) [99,100,105,106] or non-functionalized polymers like polyethylene (PE) [102,103] can only be cationized by transition metal ions in their l-t oxidation state. [99,100] The formation of evenly spaced oligomer ion series can also be employed to establish an internal mass calibration of a spectrum. [122]... 

Because frequency can be so precisely measured, the exact mass of an ion can be determined very accurately in the FTMS experiment (8). Typically, low parts-per-million accuracy can be achieved in the presence or even in the absence of an internal mass calibrant (13). In addition, a high degree of mass accuracy can be maintained for days without recalibration provided that the magnetic field remains stable. More detailed information on the theory of FTMS (1, 16, 28, 31-33) and the principles of Fourier transforms applied to spectroscopic techniques (9, 34) may be found in the literature. 

Figure 1 5 MALDI spectrum from a 2-D gel spot excised from a human proteomic study in which the corresponding spectrum of the cathepsin D precursor could be identified after using SMEC micropreparation sample preparation followed by elution and spotting onto the MALDI target plate and MALDI analysis. The peptide mass fingerprinting revealed the identity of the protein using the Mascot bioinformatic software and the Swissprot protein database. The ( ) indicates the peptide masses corresponding to the cathepsin D precursor, and (T) the trypsin peptide fragments that were used for internal mass calibration.

An internal mass calibration is generally needed to achieve mass measurement accuracy of 5 to lOppm with a Q-TOF MS analysis [62-64]. Internal calibration is based on mixing one or several internal standards or calibrants of known molecular weight with the analyte and then using the known masses to calibrate the mass measurements of unknowns that coexist in the sample mixture. 

Photolytically degraded PBO was re-examined by GC-EIMS at a higher mass spectrometer resolution (5000) with PFK in the mass spectrometer source as an internal mass calibrant. This procedure permits the accurate mass measurement of molecular and key fragment ions and hence the determination of their elemental compositions and fragmentation pathways. This information, along with the molecular weight information (GC-CIMS) and the low-resolution GC-EIMS spectrum, allows a structure to be proposed for each of the degradation compounds observed. 

FIGURE 10.4 Matrix-assisted laser desorption mass spectra of (a) peptide fragments produced by endoproteinase Lys-C digestion of melittin, (b) Endo Lys-C fragments isolated by immunopre-cipitation with mab 83144, (c) chymotrypsin fra ents of melittin, and (d) chymotrypsin fragments of melittin immunoprecipitated with antimelittin antibody mab 83144. Peaks labeled with an asterisk are dynorphan A,.,3 added as internal mass calibrant, and peaks labeled II, 12, and I are impurities. Reprinted with permission from reference 10. 

Fig. 14.13. Liquid chromatograms (from top) by photodiode array detection, HC and RIC (m/z 401) from LC-ESI-MS, and accurate mass measurement (bottom) of the unknown impurity based on a one-point internal mass calibration. Adapted from Ref. [27] by permission. Elsevier Science, 2001.

The internal mass calibration process is performed by the instrument control in the background without being noticed by the operator. In particular, it provides superior stability especially for high sample throughput with extended runtimes. 

For GC-HRMS (high mass resolution) systems, an internal mass calibration (scan-to-scan) for accurate mass determinations by control of the data system is employed. At a given resolution (e.g., 10 000), a known reference is used which is continuously leaked into the ion source during analysis. The analyser is positioned on the exact mass of the substance ion to be analysed relative to the measured centroid of the known reference. At the beginning of the next scan, the exact position of the centroid of the reference mass is determined again and is used as a new basis for the next scan (see Chapter 2.3.4.3 Lock-Plus-Cali Mass Technique). 

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