External mass calibration

All mass spectrometers require mass calibration before they are put to use. However, proper procedures and the number of required calibration points may largely differ between different types of mass analyzers. Typically, this necessitates several peaks of well-known m/z values evenly distributed over the mass range of interest. These are supplied from a well-known mass calibration compound or mass reference compound. Calibration is then performed by recording a mass spectrum of the calibration compound and subsequent correlation of experimental m/z values to the mass reference list [33,49,50]. Usually, this conversion of the mass reference list to a calibration is accomplished by the mass spectrometer s software. Thus, the mass spectrum is recalibrated by interpolation of the m/z scale between the assigned calibration peaks to obtain the best match. The mass calibration obtained may then be stored in a calibration file and used for future measurements without the presence of a calibration cotipound. This procedure is termed external mass calibration. 

Note The numerous ionization methods and mass analyzers in use have created a demand for a large number of calibration confounds to suit their specific needs. Therefore, mass calibration will variously be addressed at the end of 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 vary from mediocre 0.5 u to perfect 10 u. 

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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. 

External mass calibration was used by peak centroiding at the 80% level, except where otherwise indicated. Sample concentrations are expressed as pmole/mm of crystallized material, in order to account for the larger spread of the sample spots on PE ( 4mm) compared to the steel stages ( 1.5mm). 

FIGURE 10.7 Full-scan accurate mass spectrum of nefazodone (m/z 470) acquired in the Orbitrap mass spectrometer using external mass calibration. The expanded spectrum showed the low-intensity product ions. Product ion assignment resulted from predictive fragmentation using Mass Frontier 4.0. 

IDMS is based on measurements of masses and isotope ratios only. Some important advantages, compared with other calibration strategies, such as external calibration and standard additions, are that instrumental instabilities such as signal drift and matrix effects will have no influence in the final concentration in the sample, high accuracy and small measurement uncertainties are enabled, possible loss of substance of the isotope-diluted sample will have no influence on the final result and there is no need to resort to an external instrumental calibration or standard additions to the sample. 

External Calibration When mass calibration is conducted in an entirely separate exercise from analysis of an unknown. External calibration can be performed infrequently, avoiding the potential problem of simultaneous analysis of calibrant and unknown (direct interferences, suppression, etc.). 

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. 

External standard calibration is used if the changes in the analytical system that may occur from the time the instrument has been calibrated to the time the sample analysis is completed are negligible. These changes are assumed to produce an insignificant error that is built into the daily calibration verification acceptance criteria. The response (calibration) factor of the external standard calibration is calculated according to Equation 1, Appendix 22, and it is measured in concentration or mass units (or their inverse). 

A pair of elevated anchors with hydrophilic top surface allowed for sample and standard deposition in very close proximity (on the anchors), and thus improved mass calibration with an external standard. The approach actually mimicked an internal standard mass calibration procedure and enabled the analysis of 30 pL sample solutions corresponding to 1.5 amol of angiotensin peptides. 

A low-resolution mass measurement is a simple procedure and can be performed with most of the mass spectrometric systems discussed in Chapter 3. The instrument is set up at a resolving power (RP) above 1000. At this low resolving power, the molecular ions of most organic compounds that differ by a unit mass are well separated. The mass spectrometer is first mass-calibrated with an external calibration procedure. During the calibration scan, the computer stores the peak centroid (the center of gravity) time and the area of each peak. The mass of the ion is related exponentially to the peak centroid time ... 

Calibration of peak position for accurate mass determination can be performed internally or externally to minimize systematic errors. Internal calibration can be conducted when compounds with known molecular weight (called calibration compounds or calibrants) are mixed with the sample prior to the introduction into the ion source. This calibration can be performed, for example, by adding the calibrant to the liquid-phase sample while diluting it prior to analysis. The best result is achieved when multiple calibration signals are used to interpolate the m/z of ions within the range of interest. In proteomics, a tryptic digest of albumin from horse heart is typically used as the calibrant because it covers a wide m/z range (e.g., m/z 800-3000) that is ideal for mass calibration of low- to medium-sized peptides. In external calibration, the calibrants are analyzed before the analysis of real samples. The peaks of the calibrants are used to create and set the calibration equation in the data acquisition software. This method provides less mass accuracy because the instrument condition may still vary between the calibration and analyses of real samples. However, external calibrations save time and calibration compounds, and such methods also make analyses of analytes free from interferences caused by calibrants. 

Similar to mass calibration, quantity calibration can be done internally or externally by using appropriate calibration compounds. The calibration curve covers the intensity range corresponding to the intensities of analyte peaks. In order to obtain the highest accuracy, the calibrants used in quantity calibration should have similar atomic composition, molecular structure, and mass as analytes. This requirement is because ionization efficiency depends on molecular structure, and detection efficiency is mass-dependent. In fact, ion detectors rely on secondary electrons produced by the impact of primary ions with detector surfaces, including microchannel plates, electron multipliers, and many others. 

Because an increase in resolution causes a decrease in sensitivity, it is best to operate at the lowest resolution commensurate with good results. Some instrument data systems will allow calibration with an external reference material such as perfluorokerosene and then use of a secondary reference material for the internal mass reference. Tetraiodothiophene, vaporized using the solids probe inlet, is recommended as the secondary reference. The accurate masses are 79.9721, 127.9045, 162.9045, 206.8765, 253.8090, 293.7950, 333.7810, 460.6855, and 587.5900. For a higher mass standard, use hexaiodobenzene. Because the mass defect for these internal reference ions are so large, a resolution of 2000 is ample to separate these ions from almost any sample ions encountered in GC/MS. 

In many cases when methods involve internal or external standards, the solutions used to construct the calibration graph are made up in pure solvents and the signal intensities obtained will not reflect any interaction of the analyte and internal standard with the matrix found in unknown samples or the effect that the matrix may have on the performance of the mass spectrometer. One way of overcoming this is to make up the calibration standards in solutions thought to reflect the matrix in which the samples are found. The major limitation of this is that the composition of the matrix may well vary widely and there can be no guarantee that the matrix effects found in the sample to be determined are identical to those in the calibration standards. 

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