Lock mass

As the monitored m/z values are selected to best represent the target compound, SIM exhibits high selectivity that can be further increased by high resolution SIM (HR-SIM) because this reduces isobaric interferences. [41-44] As HR-SIM requires precise and drift-free positioning on narrow peaks, one or several lock masses are generally employed although rarely explicitly mentioned. [44,45] The role of the lock mass is to serve as internal mass reference for accurate mass measurement. (Examples are given below.)... 

Example The number of m/z values to be monitored in SIM is limited. Such limitations are more severe when additional lock mass peaks have to be included in case of HR-SIM. Therefore, it is commonplace to monitor different sets of SIM traces during consecutive time windows leading to a sequence of different SIM setups during a single chromatographic separation. The quantitation of halogen-... 

In tandem MS mode, because the product ions are recorded with the same TOF mass analyzers as in full scan mode, the same high resolution and mass accuracy is obtained. Isolation of the precursor ion can be performed either at unit mass resolution or at 2-3 m/z units for multiply charged ions. Accurate mass measurements of the elemental composition of product ions greatly facilitate spectra interpretation and the main applications are peptide analysis and metabolite identification using electrospray iomzation [68]. In TOF mass analyzers accurate mass determination can be affected by various parameters such as (i) ion intensities, (ii) room temperature or (iii) detector dead time. Interestingly, the mass spectrum can be recalibrated post-acquisition using the mass of a known ion (lock mass). The lock mass can be a cluster ion in full scan mode or the residual precursor ion in the product ion mode. For LC-MS analysis a dual spray (LockSpray) source has been described, which allows the continuous introduction of a reference analyte into the mass spectrometer for improved accurate mass measurements [69]. The versatile precursor ion scan, another specific feature of the triple quadrupole, is maintained in the QqTOF instrument. However, in pre-... 

Lock Mass Similar to internal calibration. The lock mass compound is monitored during analysis of the unknown, and the mass calibration is adjusted based on the comparison of the measured m/z and the theoretical m/z for the lock mass compound. If multiple lock mass compounds are used across the m/z range, the process effectively becomes internal calibration. Lock mass compound(s) can be introduced into the LC-MS source via a tee into the LC flow or sheath liquid inlet or dedicated sprayer. 

As shown in Fig. 4.8, the mass difference between the two metabolites is 36.4 mDa and a mass spectrometer with a resolving power of at least 9500 (M/AM = 344/ 0.0364 = 9450) is required to separate between these two metabolites. MS/MS fragmentation combined with accurate mass measurement is the preferred method for structural elucidation of metabolites, especially when it can help to correlate the elemental composition determined in the MS mode. In the example shown in Fig. 4.8, exact mass measurements of the precursor ions are used as lock mass to measure the exact mass of each fragment ion. Exact mass measurements of the fragment ion at m/z 226 help to narrow down the sites of modifications and allows one to distinguish between rabeprazole-sulphide and rabeprazole-aldehyde. 

All TOF mass spectrometers measure accurate mass by reference to standard substances, otherwise known as lock mass. This is an internal reference which is introduced via the ion source by means of a second sprayer system (Fig. 4.11) or coinfused with the sample using a single sprayer (Williams et al., 2006). However, the latter method is not preferred due to dilution of analyte with reference compound and issues of suppression of ionization (Herniman et al., 2004). The reference compound... 

Figure 4.11. Diagram showing the use of Lockspray to introduce a lock mass solution into an ESI source of a Q-TOF mass spectrometer.

Certain software algorithms such as the one mentioned previously uses the high-quality exact mass data from the high-resolution TOF systems to report calculated elemental compositions within the results browser. Good scientific practice mandates the use of an internal reference or lock mass to obtain valid exact mass measurements (sub-5-ppm). Exact mass measurement gives greater confidence in the confirmation of expected metabolites and allows the prediction of the elemental composition of unknowns. The Q-TOF mass spectrometer can be set up to automatically acquire exact mass LC-MS and exact mass MS-MS data to within the recommended mass accuracy guidelines of 5 ppm. 

The lock mass (LM) option can be used as a substitute for a more frequent calibration. Post column infusion of a known component can be utilized so that the lock mass ion is present in every scan. Lock mass mode accurate mass measurements are also possible for MS/MS experiments, where the lock mass ion is not present in... 

TABLE 5.3. Accurate Mass Measurements for a set of 30 Pharmaceutical Compounds Collected in Positive Ion Mode (Experimental Error in ppm Shown in Column 4), Negative Ion Mode (Experimental Error in ppm Shown in Column 5), and with Pos/Neg Switching (Data Shown in Columns 6 and 7, Respectively) Using a Lock Mass (LM) for Each Polarity, Reserpine, m/z = 609 for Positive Mode and Hippuric Acid, m/z 178 for Negative Mode... 

With some instruments, the instability of the analyser is such that there is a drift in mass on relatively short times, say less than 1 day. When this drift is large, the use of an internal standard or a dual source to inject ions alternately from the sample and from the standard is needed. The first solution increases the number of ions in the analyser, and thus the importance of the space charge effect. The second increases the acquisition time. Frequent recalibration of the spectrometer may also be required. If the instrument is stable, a lock mass can be used either from a compound added to the sample or from a known ion from the solvent or the background. 

A dual-iidet ESI source, the LockSpray , was introduced by Waters, based on the rotating aperture of the MUX-source, for the co-introduction of a reference compoimd to act as a lock-mass for accurate-mass determination [65-66]. Dual-inlet devices to introduce a lock-mass compound have also been reported for sector [67] and Fourier-transform ion-cyclotron resonance MS (FT-ICR-MS) instruments [68]. 

TOF and Q-TOF instraments are freqnently applied in metabolism studies. The identity of the epothilone B metabolites fonnd by precnrsor-ion analysis (Figure 10.5, Ch. 10.4.2) was confirmed using accnrate-mass determination on a LC-TOF-MS instmment [29]. Some other examples are the characterization of metabolites of moclobemide and remikiren [32], the identification of ketobemidone Phase-I and Phase-II metabolites [33], and the identification of in vitro metabolites of ethoxidine [34]. The nse of a five-channel multiplexed ESI interface (four chaimels for parallel LC-MS and one channel for lock-mass componnd infusion) on a Q-TOF instrument was recently described to speed np metabolite identification and to enhance the efficient nse of the costly instrument [35]. 

The lubricity theory explains the resistance of a polymer to deformation. Stiffness and rigidity are explained as the resistance of intermolecular friction. The plasticizer acts as a lubricant to facilitate movement of macromolecules over each other, thus giving the resin an internal lubricity. The gel theory is applied to predominantly amorphous polymers. It proposes that their rigidity and resistance to flex are due to an internal three-dimensional honeycomb structure or gel. The spatial dimensions of the cell in a brittle resin are small because their centers of attraction are closely spaced and deformation cannot be accommodated by internal movement in the cell-locked mass. Thus, the elasticity limit is low. Conversely, a thermoplastic or thermosetting polymer with widely separated points of attachment between its raacroraolecules is flexible without plasticization. 

The most common use of batch inlets is to introduce a controlled flow of compounds for calibrating the mass scale. Frequently used calibration compounds include perfluorotributylamine (FC-43, heptacosa) and perfluorokerosene (PFK) both are effective with electron ionization (El) but give limited responses in chemical ionization (Cl). If used for calibration in Cl, or to provide lock masses (Section 3.1.1), the concentration of the reagent gas must be reduced. This reducation somewhat compromises the effectiveness of the Cl process. 

Mass calibration establishes the mass scale by determining the relationship between a series of miz values, covering the mass range of interest, and the operational parameters that determine how a mass spectrum is collected, e.g., scanning of voltages on a set of quadrupole rods, or measurement of the flight times of ions in a TOF analyzer. Figure 3.1 illustrates a series of calibration ions as well as ions for an unknown and a lock mass. 

For accurate mass measurements the unknown miz are determined with respect to a lock mass to account for both tntra- and inter-run variability. 

The lock mass does not have to be one of the calibrant ions, but it has to be a compound with a known empirical formula. 

FIGURE 3.1 Mass scale calibration and the use of a lock mass. 

Many modem instruments, e.g., time-of-flight (TOF) systems, can be used to determine accurate masses however, it is often questionable whether such numbers are important enough to justify the necessarily stringent calibration, lock mass, and concentration requirements, as well as the more advanced training of users. This is particularly true when MS is moved to the beginning of the analytical sequence, when knowledge of the nominal mass is often sufficient. For instance, accurate mass measurement is often irrelevant when the objective is to synthesize a compound of... 

The amount of energy released by the incident beam is sufficiently high in the vicinity of the point of impact that sample molecules are ionized by proton transfer from a protonated matrix. Typically, the spectrum obtained shows cluster ions formed from the matrix. In particular, the positive ion spectrum of glycerol (mw 92 u) shows a series of ions 92 +l where n can be 1 up to 12 or more. This spectrum can be used to calibrate the mass spectrometer and also provide internal lock masses for accurate mass measurement, described in applications below. 

Lock mass reference leucine enkephalin infused with a lock-spray of 5 pL/min. 

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