Calibration, mass accuracy

The LTQ-Orbitrap has resolution and mass accuracy performance close to that of the LTQ-FTICR. As shown in Table 5.3 (column 4), LTQ-Orbitrap accurate mass measurements, using external calibration, for a set of 30 pharmaceutical compounds resulted in less than 2.3 ppm error. The data were acquired with a 4-min, 1-mL/min-flow-rate, positive-mode LC-ESI-MS method where all measurements were performed within 5h from mass calibration. Mass accuracies below 2-3 ppm, and often below 1 ppm, can be routinely achieved in both the positive- and negative-ion mode (Table 5.3, columns 4 and 5). The long-term mass stability of the LTQ-Orbitrap is not as consistent as observed for the LTQ-FTICR-MS, and the Orbitrap requires more frequent mass calibration however, mass calibration is a routine procedure that can be accomplished within 5-10 min. Figure 5.7 displays a 70-h (external calibration) mass accuracy plot for three negative ions collected with a LTQ-Orbitrap where the observed accuracy is 2.5 ppm or better with little mass drift for each ion. Overall, for routine accurate mass measurements on the Orbitrap, once-a-week calibration (for the desired polarity) is required however, considering the ease of the process, more frequent external calibration is not a burden. 

Senko, M., Zabrouskov, V., Lange, O., Wieghaus, A., and Homing, S. (2004). LC/MS with external calibration mass accuracies approaching 100 ppb. In Proceedings of the 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN. 

Most FTICR mass spectrometers use superconducting magnets, which provide a stable calibration over a long period of time. Although some mass accuracy can be obtained without internal calibrant, mass accuracy and resolution are inversely proportional to m/z, and the best accurate mass measurements require an internal calibrant. Unlike the quadrupole ion trap, the FTICR mass spectrometer is not operated as a scanning device. 

Mass spectrometers are usually characterized by their internal and external calibration mass accuracy, or how closely the measured mass matches the theoretical mass. It is usually reported in Da or in parts per million, but as mass accuracy varies across the spectrum in many mass spectrometers it must also be reported at a parhcular m/z value. Every mass spectrometer has a characteristic mass accuracy capability, but achieving this best-case limit usually requires careful control of the instrumental parameters and ensuring that the ion source and ion ophcs are clean. MicroChannel plate... 

TOF mass spectrometers are very robust and usable with a wide variety of ion sources and inlet systems. Having only simple electrostatic and no magnetic fields, their construction, maintenance, and calibration are usually straightforward. There is no upper theoretical mass limitation all ions can be made to proceed from source to detector. In practice, there is a mass limitation in that it becomes increasingly difficult to discriminate between times of arrival at the detector as the m/z value becomes large. This effect, coupled with the spread in arrival times for any one m/z value, means that discrimination between unit masses becomes difficult at about m/z 3000. At m/z 50,000, overlap of 50 mass units is more typical i.e., mass accuracy is no better than about 50-100 mass... 

Other techniques for mass measurement are available, but they are not as popular as those outlined above. These other methods include mass measurements on a standard substance to calibrate the instrument. The standard is then withdrawn, and the unknown is let into the instrument to obtain a new spectrum that is compared with that of the standard. It is assumed that there are no instrumental variations during this changeover. Generally, this technique is less reliable than when the standard and unknown are in the instrument together. Fourier-transform techniques are used with ion cyclotron mass spectrometers and give excellent mass accuracy at lower mass but not at higher. 

Principles and Characteristics Mass spectrometry can provide the accurate mass determination in a direct measurement mode. For a properly calibrated mass spectrometer the mass accuracy should be expected to be good to at least 0.1 Da. Accurate mass measurements can be made at any resolution (resolution matters only when separating masses). For polymer/additive deformulation the nominal molecular weight of an analyte, as determined with an accuracy of 0.1 Da from the mass spectrum, is generally insufficient to characterise the sample, in view of the small mass differences in commercial additives. With the thousands of additives, it is obvious that the same nominal mass often corresponds to quite a number of possible additive types, e.g. NPG dibenzoate, Tinuvin 312, Uvistat 247, Flexricin P-1, isobutylpalmitate and fumaric acid for m = 312 Da see also Table 6.7 for m = 268 Da. Accurate mass measurements are most often made in El mode, since the sensitivity is high, and reference mass peaks are readily available (using various fluorinated reference materials). Accurate mass measurements can also be made in Cl... 

Tables 6.27 and 6.31 show the main characteristics of ToF-MS. ToF-MS shows an optimum combination of resolution and sensitivity. ToF-MS instruments provide up to 40000 spectra s-1, a mass range exceeding 100000 (in principle unlimited), a resolution of 5000, and peak widths as short as 200 ms. This is better than quadruples and most ion traps can handle. Unlike the quadrupole-type instrument, the detector is detecting every introduced ion (high duty factor). This leads to a 20- to 100-times increase in sensitivity, compared to QMS used in scan mode. The mass range increases quadratically with the time range that is recorded. Only the ion source and detector impose the limits on the mass range. Mass accuracy in ToF-MS is sufficient to gain access to the elemental composition of a molecule. A single point is sufficient for the mass calibration of the instrument. ToF mass spectra are commonly calibrated using two known species, aluminium (27 Da) and coronene (300 Da). ToF is well established in combination with quite different ion sources like in SIMS, MALDI and ESI.

SEC-ESI-FTMS combines the size separation based technique of SEC with one of the most powerful mass spectrometric techniques of FTMS offering high mass accuracy (ppm), ultrahigh resolving power (>10(i) 6) and the capability to perform tandem mass spectrometry. The technique enables generation of oligomer elution profiles, which can be used for accurate calibration of standard SEC data. Coupling of SEC to ESI-MS is further described in ref. [710],... 

With the FT-ICR mass spectrometer it is possible to obtain high mass accuracy (about 1-5 ppm calibrated externally, 0.5-1-0 ppm if calibrated internally here an external calibration has been carried out using a tune mix containing compounds with m/z from... 

The mass accuracy is highly dependent on the mode the instrument is operating in. In the reflector mode, with time-lag focusing, the best MALDI-TOF and oa-TOF instruments are capable of achieving <5 ppm with internal standards, provided that the isotopes are resolved. In many cases it is not possible to add internal calibrants, and then the error in mass accuracy is often increased to 50-100 ppm. Operation of an instalment in a linear mode will typically decrease the mass accuracy. 

Quadmpole analyzers have generally been considered to give poor mass accuracy. Recently, however, with better machined parts and better electronics, commercially available instruments can perform quite well. A mass accuracy <5 ppm can be obtained with internal calibrants. 

The orbitrap mass accuracy is better than all quadmpoles and TOF instruments just right after the FTICR and sector instruments, that is, around 2 ppm with internal calibration [248]. 

A mass calibration for FTICR analyzers with superconducting magnets is very stable and is valid for many days for normal applications. Mass accuracy < 1 ppm can be obtained over a fairly wide mass range. Unique elemental composition can be determined for masses over 800 Da [262]. Recently, 0.1 ppm mass accuracy, which required a mass resolving power >300,000, has been achieved for several thousand peaks by a 14.5 T instrument [263] and commercial instruments with mass accuracy <0.2 ppm are available. As with the orbitrap (see Section 2.2.5) the frequency is... 

It is often neglected that the first step of de novo sequencing is data acquisition. The quality of the spectrum or spectra used for sequencing is the most critical parameter of the entire procedure. First of all, the mass spectrometer should be well calibrated and tuned. If it can operate in different modes, the one with the highest possible mass accuracy and resolution should be applied. If the experimenter has more spectrometers to choose from, the one with the highest mass accuracy and resolution should be used, provided it shows good fragmentation efficiency. 

So far, the concepts of exact mass, mass accuracy and resolution have been introduced without considering the means by which accurate mass measurements can be realized. The key to this problem is mass calibration. Resolution alone can separate ions of different m/z value, but it does not automatically include the information where on the m/z axis the respective signals precisely are located. 

Note The numerous ionization methods and mass analyzers in use have created a demand for a large number of calibration compounds to suit their specific needs. Therefore, mass calibration compounds will occasionally be addressed later in the chapters on ionization methods. It is also not possible to specify a general level of mass accuracy with external calibration. Depending on the type of mass analyzer and on the frequency of recalibration, mass accuracy can be as high as 1 mmu or as low as 0.5 u. 

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. 

How often an LC-MS should be calibrated depends on the mass accuracy required. For example, instrument calibration should be verified daily when performing accurate mass measurements of peptides and proteins. However, the quantitative analysis of small molecules requires less frequent calibration. 

In ICP-MS a multi-element tuning solution is applied for the mass calibration of mass spectra. Figure 6.3 shows the mass spectrum for phosphorus determination at m/z = 31. In this case, the mass calibration was performed with the aid of a solution of a phos-phorus/sulphur mixture. From the known masses of the isotopes of the atomic ions, the masses of the polyatomic ions occurring were determined and identified in accordance with the isobaric polyatomic ions (15N160+ and 14N16OH+). Mass accuracy is the deviation of experimental determined mass of an atomic, polyatomic, cluster, molecular or fragment ion from the exact mass of species expected. Polyatomic, cluster and molecular ions were calculated from the atomic masses (see Appendix I), the mass accuracy is usually presented in ppm. 

One of the most attractive features of the LTQ-FT is the outstanding mass accuracy achieved with external calibration in both MS and MS/MS modes. The generally... 

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. 

Using a horizontal 4.7 Tesla cryoshimmed supercon magnet with a bore diameter of 15 cm, we have obtained two-parameter mass calibrations with absolute mass accuracies of better than 1.5 ppm without, and better than 0.4 ppm with an internal calibrant over a mass range from 18 to 502 amu (10). Absolute accuracies with an internal calibrant are slightly better because the calibrant and the unknown compound are measured under identical physical conditions, in particular with the exact same number of ions in the cell, resulting in identical space-charge shifts (2). 

Calibrate the spectra to ensure reliable mass accuracy. Generate a calibration equation using calibrants that cover the mass range of interest, and then match the calibrant and sample matrix. External mass calibration can subsequently be applied to the relevant spectra. 

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