HIGH ACCURACY SELF-CALIBRATION METHOD FOR HIGH RESOLUTION MASS SPECTRA

Boris Kozlov1, Vasiliy Makarov2, Keith Richardson1, Jeff Brown1 1. Waters, Wilmslow, UK; 2. MS Consulting, Bar, Montenegro.

SELF-CALIBRATION BASED ON COMPREHENSIVE GRID OF ELEMENTAL

COMPOSITIONS.

RESULTS AND DISCUSSION EXPERIMENT.

Normally, in practical cases mass

The method was applied to LC-MSe data of urine samples acquired with the rate 10 spec-tra per second by multi-reflecting ToF MS with resolving power 200 K.

FINDING THE CORRECT CALIBRATION FOR AN ARBITRARY TOF MS SPECTRUM We monitor a persistent peak of m/z=279.0933 Da in the chromatograms under consideration. Due to unavoidable instabilities of power supplies in sub Hz range at low ppm level, the peak centroid is jumping from spectrum to spectrum in frames +-1ppm with RSD about 0.5 ppm (in mass scale). After the application of procedure of self-

OVERVIEW

PURPOSE: A novel de-novo method of calibration of

high resolution mass spectra is presented

METHODS: A combinatorially generated matrix of

INTRODUCTION

Modern high-resolution mass spectrometers can provide

mass accuracy at the sub-ppm level. The best results

can be achieved using internal calibration. If internal

calibration is not used, the accuracy of peak

identification is limited by stability of parameters of the

mass spectrometer, and by the influence of external

fields and temperature .

An important advantage of high-resolution mass

spectrometers is clear differentiation of peaks with

The correct group of assignments is to be the most populated, as all clusters related to substitutions of any particu-lar elemental group are projections of only part of the main group.

Incorrect groups of assignments can be recognized by an obvious bias of elemental compositions (as an example, all assignments in the group contain nitrogen).

Adjustment of only a and t calibration coefficients for ToF MS spectrum results in coincidence of the choice of cor-

Normally, in practical cases mass spectra contain a lot of peaks related to organic species with general chemical formula

CnCHnHOnONnN We used this formula together with chemical restrictions on C valence bonds to constrain the ratios between numbers of atoms. The resulting set (matrix) of precise mass values, was used for best fitting with meas-ured times of flight by adjustment coefficients a0 and t0 in the calibration formula (1). The coefficients are adjusted separately for every single spectrum of the chromatogram.

resolving power 200 K. Known metabolites were added to allow an independent estimate of the accuracy of mass measurement. The accuracy was also esti-mated by monitoring variation of the measured mass of a persistent peak of triphenyl-phosphine oxide (279.0933 Th)

Figure 7. Plot of mass differences after completion of the self-calibration procedure. Groups of false assignments are shifted by m, corresponding to

known substitution of groups of elements or isotopes. Vertical axis: m (Th), horizontal axis: m/z (Th)

XIC of 2 pg tianeptine 5 mDa window

TIC urine sample U4

0.5 ppm (in mass scale). After the application of procedure of self-calibration, variation of the peak centroid position in mass scale decreases to RSD~0.1 ppm.

Figure.10. Top: initial oscillation of the measured mass of a persistent

peak (triphenylphosphine oxide). Bottom: the same peak mass variation

after application of self-calibration procedure to every spectrum.

The improvement of peak position stability is due to the fact that the fitted calibration coefficients compensate for those minor variation in every spectrum. Note that this compound does not appear in the calibration grid as it contains phosphorous. Note also that attempting to smoothly

METHODS: A combinatorially generated matrix of

elemental compositions is matched to peak detected

data, and the matches are used to fix two calibration

parameters

RESULTS: Sub 100ppb mass accuracy is demonstrated Figure 2. Parameters of the

LC method

spectrometers is clear differentiation of peaks with

different elemental composition over a wide mass range.

This makes it feasible to use a grid of masses of all

reasonable elemental compositions to correct the mass

calibration.

The best results can be achieved for high resolution

spectra that are not subjected to local distortions of

mass scale related to high signal amplitudes or other

nonlinearities [1].

We applied the new self-calibration approach to LC-MS

spectra acquired with a high-resolution time-of-flight

mass spectrometer at the rate of 10 spectra/s.

CONCLUSION

Figure 8. Left: Correct choice. Group of correct assignments is marked by red star. Right: Assigning the wrong cluster (CH2—N substitution) would re-

sult in curvature of the group in the ( m vs m ) plot

Adjustment of only a0 and t0 calibration coefficients for ToF MS spectrum results in coincidence of the choice of cor-rect group of assignments with smallest spread of peak-to-grid distances. Choice of the wrong group will result in some mass-dependent deviation (second order curvature of the calibration fit).

HISTORICAL METHOD OF SELF-CALIBRATION IN TOF MS USING INTEGER MASSES

XIC of 2 pg tianeptine 20 mDa window

Figure 3. One of examined chromatograms (Survey of non-fragmented

ions). High resolution and good calibration help to identify components.

Figure 5. An example of a complex peak of mass of a “whole mass” 123

fitted to the grid of precise masses of the generated set of elemental com-

positions.

PLOT OF ALL DISTANCES BETWEEN PEAKS AND

GRID POSITIONS .

a0=1.849335E-10 t0=212

a0=1.849200E-10 t0=137

Note, to keep plots “horizontal” (constant offsets for all groups) one needs to adjust two parameters (a0 and t0)

as it contains phosphorous. Note also that attempting to smoothly interpolate the calibration between spectra would make the results worse.

Figure.11. Fitted coefficients are following the peak position oscillations

m/z

Err

or

(Da)

Number of hits

m/z

Err

or

(Da)

Number of hits

Table.1. Measured masses for known compounds (60 pg and 2 pg

concentrations) following application of the self-calibration procedure.

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References

1. Boris Kozlov; Richard Denny, Yuriy Khasin, Sergey Kirillov; Vasiliy

Makarov, Anatoliy Verenchikov, John Hoyes; Enhanced mass

accuracy in multi-reflecting TOF MS. ASMS 2017

CONCLUSION

Application of the method to high resolution LC-MS data of methabolomic samples shows that better than 0.1 ppm mass accuracy can be achieved without any use of known external or internal calibrants.

This self-calibration eliminates the requirement for external introduction of lock-masses, and alleviates the need for thermal stability of power supplies and mechanical parts of an analyzer.

Due to the known non-linear dependence of m/z over the time of flight, good fit (calibration parameters a0, t0) can be achieved only for correct assignments of whole m/z values to the peaks. Inaccuracy of the method results from mass defects (masses of ions are not precisely proportional to number of protons and neutrons in their nuclei)

Figure 1. An example of finding correct mass calibration by fitting peaks

to a whole-mass grid.

Figure 4. Top: an example mass spectrum (at RT=100s ).

Bottom: Representation of the same mass spectrum as mass-defects plot-

ted against mass to illustrate the plurality of peaks in experimental spec-

trum..

Figure 9. Distribution of mass-differences between measured peaks and the grid of elemental compositions. The points, forming the central group

(peak) are considered to present a set of correct assignments, and are used for precise fitting of mass calibration of the current spectrum.

Top: Histogram for correct assignment (Fig. 8 left). Bottom: Histogram for the same data with wrong group choice. (Fig. 8 right)

Figure 6. Top: Plot of all mass differences between measured peaks and

available values of the grid of elemental compositions, where masses are

estimated using some initially introduced calibration coefficients a0 and t0.

Bottom: Zoom to ±10 mDa window.

Vertical axis: m (Th), horizontal axis: m/z (Th)

correspondingly. Use of a three-parameter fit (parabola) may result in wrong t0 value and loss of calibration linearity.

(1)