Accurate Mass and TIP^TM: Two Dimensions for Elemental Composition Determination: microTOF^TM Featuring Unique TIP^TM

The ESI-TOF mass spectrometers micrOTOF and micrOTOF focus do not only provide accurate mass measurements but additionally describe the True Isotopic Pattern TIP^TM. For molecular formula generation this unique combination significantly reduces the number of possible formula candidates and the elemental composition of compounds with molecular weights far higher than 500 Da can unequivocally be determined.

Introduction

Mass spectrometry is widely used for the determination of the elemental composition in drug development, metabolomics and discovery chemistry applications. Last generation ESI-TOF mass spectrometers provide a mass accuracy better than 5 ppm which is usually adequate for an unequivocal formula assignment for molecules up to 300 Da. If molecules larger than 500 Da have to be analysed, multiple formula suggestions usually conform to the determined molecular weight within the specified mass accuracy even when additional chemical knowledge is considered.

The micrOTOF^TM was developed for accurate mass measurements of unknown compounds. Additionally to a mass accuracy better than 3 or 5 ppm (internal and external calibration, respectively), the unique True Isotopic Pattern (TIP^TM) considers the correlation between measured and theoretical isotopic pattern, being presented as Sigma-FIT^TM.1 This unique combination results in a significant reduction of the number of potential formulae (Figure 1).

This note presents the elemental formula assignment of ginsenosides from a commercially available ginseng extract.

Experimental
Separation was performed by using an Agilent 1100 HPLC system equipped with binary pump and solvent degasser, autosampler and column compartment. The output of the HPLC separation was introduced into the ESI source of the Bruker Daltonic MicrOTOF time-of-flight LC-MS system, operated at a resolving power of 10 000 (FWHM) in negative-ion mode.

A commercial available ginseng tonic preparation was diluted 1:100 with water. The HPLC separation made use of a Waters 3 mm x 100 mm Xterra MS 3.5 µm 18 column with a flow-rate of 0.25 mL/min. Solvent A was 0.1% formic acid in water and solvent B was acetonitrile. The solvent composition was varied from 10% B to 95 % B in 30 minutes. 10 µL of the diluted sample was injected.

Results
The commercial ginseng extract was obtained from Panax ginseng C.A. Meyer (Asian ginseng). The ginsenosides in the extract were separated and analysed by LC-ESI-TOF mass spectrometry. We were able to assign 13 different ginsenosides eluting between 17 min and 28 min (Figure 2(a)). The proceeding for the elemental composition determination will be presented exemplary for the ginsenoside #4 eluting after 23.2 min being one of the compounds with the highest molecular weight in the studied extract.

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The ginsenoside #4 gave a singly negatively charged ion at m/z 1153.5982 in ESI negative mode. This information was used in the "Generate Molecular Formula" editor of the post-processing software DataAnalysis^TM for the calculation of possible formulae. 171 candidates were found within the specified mass error tolerance of 5 ppm using external calibration. In a second calculation step the rings plus double bounds were set to a maximum of 20. Additionally, it was assumed that the electron configuration of the ion is even which is consistent with electrospray ionization. Thus, the number of candidates was reduced to 30. We expected that the compound of interest is a ginsenoside. All known ginsenosides do not contain any nitrogen. Therefore, the maximum number of nitrogen atoms was set to 0 in the final calculation step resulting in two remaining candidates (Figure 2(b)).

The isotope pattern of both possible formulae was visually consistent with the measured isotope pattern. Therefore, the formula C₆₂H₈₉O₂₀ seemed to be more probable since it showed the better mass fit. However, the ranking according to the Sigma- Fit resulted in the correct formula C₅₅H₉₃O₂₅ being consistent with the formate adduct of the ginsenoside R_b1 already described in literature².

The following ginsenosides were additionally detected in the extract: Rg₁, R_e, R_f, mR_b1, R_c, mR_b2, R_b3, R_b2, R_g2, R_d, mR_d and mR_d-iso.

Conclusions
Accurate mass measurements within a mass error in the low ppm range are insufficient for unequivocal molecular formula assignment for compound larger than approx. 300 Da. The consideration of the correlation between measured and theoretic isotope distribution made possible by Bruker Daltonics unique TIP^TM adds a second decision dimension for elemental composition determination. This results in unequivocal formula suggestions even for compounds larger than 1000 Da.

References

1. Bruker Daltonics Technical Note # 8, New Technology for Detection of True Isotopic Pattern in Orthogonal TOF Mass Spectrometry.
2. T.W.D. Chan et al., Anal. Chem., 72(6), 1281-1287 (2000)

Source: RECENT APPLICATIONS IN LC-MS LC·GC Eur., 17(11a)38-39 (2004)