Anal. Chem. 1999, 71, 5557-5562
Technical Notes
Discovery of Quasi-Molecular Ions in Electrospray Spectra by Automated Searching for Simultaneous Adduct Mass Differences Ekkehard Go 1 rlach* and Ramsay Richmond†
Core Technology Area Analytics Unit, Novartis Pharma AG, CH-4002 Basel, Switzerland
Our high-throughput flow injection analysis mass spectrometry (FIA-MS) system using electrospray ionization steers the majority of ion current into quasi molecular ion and adduct m/z positions. One advantage of this dawned on us, i.e., that adducted electrospray spectra are amenable to automated quasi molecular ion discovery by searching for the simultaneous occurrence of adduct mass differences. Therefore, an adduct scanning algorithm was developed, given a graphical user interface, and then incorporated into a Visual Basic application originally developed for reporting of high-throughput FIA-MS results. In any interpretation of a mass spectrum, the first step is usually the manual assignment of molecular weight. Since methane positive chemical ionization (CI) was first reported in 1966, it has been extensively used in molecular weight determination of molecules under 800 a.m.u.1-3 The principal reason for its popularity was that the quasi molecular ion [M + H]+ generally had greater stability than the molecular ion [M]+ in electron impact ionization. Prominent adduct ions exist, i.e., [M + C2H5]+ and [M + C3H5]+ and to a lesser extent the [M + CH5]+ species.3-4 Occasionally observed ions include the [2M + H]+, [2M + CH5]+, [2M + C2H5]+, and [M + C3H5]+ species.5 As stated in ref 6, “initially, these adduct ions appear to confuse the spectrum, but actually they have diagnostic value in the determination of M” as their simultaneous occurrence allows them to be used as powerful pointers for the assignment of molecular weight. By analogy with methane positive ion CI, adducts in thermospray7 * Corresponding author. † Corresponding author on analytics. (1) Munson, M. S. B.; Field, F. H. J. Am. Chem. Soc. 1966, 88, 2621-2630. (2) Munson, M. S. B.; Field, F. H. J. Am. Chem. Soc. 1966, 88, 4337-4345. (3) Wilson, M. S.; Dzidic, I.; McCloskey, J. A. Biochim. Biophys. Acta 1971, 240, 623-626. (4) Fales, H. M.; Milne, G. W. A.; Vestal, M. L. J. Am. Chem. Soc. 1969, 91, 3682-3685. (5) Maryanoff, C. A.; Caldwell, G. W.; Chang, S. Y. Org. Mass Spectrom. 1988, 23, 129-134. (6) Leclercq, P. A.; Desiderio, D. M. Org. Mass Spectrom. 1973, 7, 515-533. (7) Volmer, D.; Preiss, A.; Levsen, K.; Wu ¨ nsch, G. J. Chromatogr., A 1993, 647, 235-259. 10.1021/ac9904011 CCC: $18.00 Published on Web 10/27/1999
© 1999 American Chemical Society
and electrospray ionization (ESI)8 are also frequently seen. The electrospray adduct ion intensities relative to the quasi molecular ion are more variable compared with the relative intensities in methane positive ion CI, but a compensatory feature for structure elucidation is that the adducts in ESI are more diverse. Unfortunately, this diversity is potentially confusing as it can obscure the quasi molecular ion. This is especially so in our high-throughput FIA-MS, as the chemists see their spectra directly on their workbench PC screens, containing no written quasi molecular ion annotation by mass spectrometrists. However the high degree of adduction seen in soft ESI, i.e., with no intentional in-source collision-induced dissociation, offers the possibility of automated quasi molecular ion discovery, by scanning a spectrum for the simultaneous occurrence of adduct mass differences. The work presented here has some similarities to the halogen isotope cluster enhancement programs reported in, refs 9-11, although our emphasis is on mass difference, and the relative ion intensity conditions are much looser. The RackViewer system developed to deliver the FIA-MS results quickly and directly to our sample-submitting chemists has been reported.12 RackViewer has four main views, specifically Overview, Spectrum, 3-D map, and Chromatogram.13-14 A movable adduct stencil developed to overlay any spectrum within the Spectrum view and serve as a quasi molecular ion discovery “scanner” has been briefly discussed.13 Here we focus on two major enhancements to this quasi molecular ion discovery tool. First, the reporting within the Spectrum view of the cumulative adduct ion current (∑ %RA) found in the movable adduct stencil. Second, the application of this movable adduct stencil linked with ∑ %RA evaluation, to automated quasi molecular ion discovery within the bird’s eye 3-D map,13 either for individual combinatorial syntheses or for contiguous liquid chromatography (LC) fractions in a 96-well rack.14 In this, putative quasi molecular ions are picked (8) (9) (10) (11) (12)
Karlsson, K.-E. J. Chromatogr., A 1998, 794, 359-366. Canada, D. C.; Regnier, F. E. J. Chromatogr. Sci. 1976, 14, 149-154. Anderegg, R. J. Anal. Chem. 1981, 53, 2169-2171. LaBrosse, J. L.; Anderegg, R. J. J. Chromatogr. 1984, 314, 93-102. Hegy, G.; Go¨rlach, E.; Richmond, R.; Bitsch, F. Rapid Commun. Mass Spectrom. 1996, 10, 1894-1900. (13) Go ¨rlach, E.; Richmond, R.; Lewis, I. Anal. Chem. 1998, 70, 3227-3234. (14) Richmond, R.; Go ¨rlach, E.; Seifert, J.-M. J. Chromatogr., A 1999, 835, 2939.
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out and color-highlighted from large slabs of MS data. A semipreparative LC fraction set of a multiple parallel combinatorial chemistry synthetic end product was used, first, to illustrate the practicability of these ideas and, second, to show the integration of these various discovery tools in a unified visual interface. 1. EXPERIMENTAL 1.1. LC Fraction Set. The combinatorial synthetic end product had an expected stoichiometric formula C31H42N4O6, with a monoisotopic molecular weight ) 566.3. From 110 mg of crude product, a 2-mg aliquot was separated by LC for subsequent FIAMS analysis.14 The LC solvent delivery system and solvent gradient has been described.14 In total, 84 fractions each consisting of 0.6 mL, representing 0.33-min intervals, were collected into Micronic 45 × 8.8 mm tubes. The tubes were evaporated to dryness and placed in a 96-well Micronics rack in positions B1-H12 and sent for FIAMS. 1.2. Sample Delivery and Preparation. The received evaporated residue in each 96-well tube was redissolved in 0.2 mL CH3CN/H2O 70:30 v/v. In parallel to the rack’s physical delivery to the MS laboratory, a Microsoft Excel CSV format file describing the sample positions and expected stoichiometric formulae was electronically deposited on a file server by the sample-submitting synthetic chemist. The MS operator then calls the Excel file, and UNIX script files automatically build the mass spectrometer analysis page method list.12-14 Each 10-µL sample injection is followed by two loop washes (10 µL CH3CN/H2O 70:30 v/v) to waste. The FIA-MS measurement duty cycle for each LC fraction is 90 seconds. Samples are usually measured in a left-to-right sequence, B1 to B12, C1 to C12, ... H1 to H12. Each LC fraction rack has a yohimbine standard curve installed in row A by the MS operator, for the sake of maintaining interrack comparability. This was made from yohimbine hydrochloride (Sigma part no. Y-3125), dissolved in 1.0 mL CH3CN/H2O 70:30 v/v.14 For LC fraction racks, the synthetic chemists often supply no expected stoichiometric formula in the accompanying Microsoft Excel CSV files. In this case, the MS operator modifies the CSV file so that the purity estimation defaults to reporting yohimbine purities for the entire rack, not just for the yohimbine standard curve inserted by the MS operator in row A. If the synthetic chemists supply a formula, then yohimbine purities are reported only for the standard curve in row A. 1.3. Mass Spectrometry. A Finnigan MAT SSQ-7000 single quadrupole mass spectrometer (San Jose, CA) was used, controlled by a DEC AXP 3000/300 workstation under the OSF/1 V3.2D operating system running the Finnigan Interactive Chemical Information System (ICIS) V8.2.1 application software. Two Ethernet interfaces connected the workstation to the SSQ-7000 and the Novartis local area network (LAN), respectively. A dedicated file service accessible by the synthetic chemists was set up on the Novartis LAN, to exchange files with the DEC AXP 3000/300 via TCP/IP (Samba). The Finnigan ICIS package controls a Hewlett Packard 1090 series II liquid chromatograph through a HP-IB interface and the autosampler via the RS-232C interface. An eight 96-well rack autosampler was used, i.e., the model HTS PAL from CTC Analytics, CH-4222 Zwingen, Switzerland. 5558 Analytical Chemistry, Vol. 71, No. 24, December 15, 1999
Figure 1. Schematic of ion current inclusion criteria in the discovery algorithm for the quasi molecular ion and the adduct ion.
The MS operating conditions in ESI positive mode were: heated capillary temperature, 220 °C; conversion dynode at 15 kV; electron multiplier at 0.9 kV, a collision induced dissociation offset of 0.0 V and a spray voltage of 4.5 kV. The scan range was 160-1560 m/z per 2 s in centroid acquisition mode. The FIA solvent was CH3CN/H2O 70:30 v/v as in ref 14. The FIA flow rate was 50 µL/min. Normally, 2 µL of sample was injected, onto a 10-µL loop. The connection between the autosampler LC valve and the exit end of the electrospray spray nozzle was as in ref 14. The head pressure for the ESI source nitrogen sheath gas was 60 p.s.i. The particular Finnigan API interface version no. was as in ref 14. A 0.001 mg/mL solution of yohimbine hydrochloride, reserpine, and cyclosporin A in CH3CN/H2O 70:30 v/v, infused at 50 µL/min was used to coarse-tune in profile acquisition. For subsequent fine tuning to accentuate the adduction characteristics of the electrospray tuning, the yohimbine [M + H + CH3CN]+/ [M + H]+ (or [M + Cl]-/[M - H]-) profile signal ratio was maximized. The RackViewer application for networked reporting of FIAMS results has been reported.12-14 The discovery algorithm was implemented in C++ (Microsoft Visual C++, version 4.2) and loaded into RackViewer as a dynamic link library. 2. RESULTS 2.1. Criteria Used for Quasi Molecular Ion Discovery. To include ion current in the quasi molecular ion discovery program and report it as ∑ %RA within the Spectrum and 3-D map views, all the following criteria (Figure 1) are sequentially met: (1a) The quasi molecular ion, e.g., [M + H]+ or [M - H]-, must have ion intensity above 1 %RA. (1b) An A + 1 ion must be present,15 at quasi molecular + (1 ( 0.2) m/z. (1c) This A + 1 ion must fulfill an intensity isotope criterium (eq 1). (1d) If there is an ion at the quasi molecular - (1 ( 0.2) m/z, it can not have greater intensity than the quasi molecular ion. (2a) For an ion to be regarded as a quasi molecular one, there must be at least one adduct with the correct ∆m/z, e.g., [M + Na]+ must have 22 ( 0.2 m/z units greater than the [M + H]+. (2b) An A + 1 ion must be present, at adduct + (1 ( 0.2) m/z. (2c) This A + 1 ion must fulfil an intensity isotope criteria (eq 1). (2d) If there is an ion at the adduct - (1 ( 0.2) m/z, it can not have greater intensity than the adduct ion.
Table 1. List of Cationic Adducts Displayed by the Adduct Stencil in the Spectrum View
Table 2. List of Anionic Adducts Displayed by the Adduct Stencil in the Spectrum View
m/z position
cation identity
m/z position
anion identity
(M + 2)/2a M+1 M+7 M + 18 M + 20 M + 23 M + 39 M + 42 M + 48 M + 64 M + 80 M + 83 (2M) + 1 (2M) + 18 (2M) + 23 (2M) + 39
[M + 2H]2+ a [M + H]+ [M + Li]+ [M + NH4]+ [2M + H + K]2+ [M + Na]+ [M + K]+ [M + H + CH3CN]+ [M + Li + CH3CN]+ [M + Na + CH3CN]+ [M + K + CH3CN]+ [M + H + CH3CN + CH3CN]+ [2M + H]+ [2M + NH4]+ [2M + Na]+ [2M + K]+
(M - 2)/2a M-1 M + 21 M + 35 M + 37 M + 79 M + 45 M + 59 M + 113 (2M) - 1 (2M) + 21 (2M) + 37
[M - 2H]2- a [M - H][M + Na - 2H][M + Cl][M + K - 2H][M + Br][M + HCO2][M + CH3CO2][M + CF3CO2][2M - H][2M + Na - 2H][2M + K - 2H]-
a The adduct in italics is not incorporated in the quasi molecular ion discovery algorithm either in the Spectrum or 3-D map views.
Criterion 1d excludes the A + 1 and A + 2 part of the quasi molecular cluster being reported as quasi molecular ions. Criterion 2d excludes other confusions, e.g., the ∆m/z between A + 1 of the [M + K]+ and the monisotopic of [M + Na]+ being interpreted as that between the monoisotopic of [M + H]+ and the monoisotopic of [M + NH4]+, or the ∆m/z between A + 1 of the [M + K]+ cluster and the monoisotopic of [M + NH4]+ being interpreted as that between the monoisotopic of [M + H]+ and the monoisotopic of [M + Na]+. As for criteria 1c and 2c, the A + 1 intensity is contributed predominately by carbon, nitrogen, silicon, and sulfur and to a lesser extent hydrogen and oxygen.15 Silicon and sulfur can be excluded as their stoichiometry in synthetic end products makes them generally unimportant in A + 1 ion contributions. Novartis’ synthetic chemistry was examined for the highest relative A + 1 content. An averaging of the relative ratios of C H N O in 420 stoichiometric formulae from five diverse combinatorial chemistries gave five ratio sets, Table 3. The highest nitrogen/carbon ratio was typified by the formula C18H29N4O2. Therefore, the A + 1 relative ion intensity at mw ) 333, will be (18 × 1.1%) + (29 × 0.015%) + (4 × 0.37%) + (2 × 0.04%) ) 21.8%. This allows the following equation to be derived: 1. %RA[A + 1]/%RA[quasi molecular] ) (deviation factor) (0.218) (ion’s m/z value)/333 However, under the soft ESI conditions used, there will be some deviation of the cluster shape from the theoretical, and therefore, a factor of 1.5 was included in the above equation. This equation was used in criteria 1c and 2c to exclude all quasi molecular or adduct ions that had greater than the expected A + 1 ion current. 2.2. Quasi Molecular Ion Discovery in Individual Spectra. This is implemented in two subviews within the Spectrum view, Figure 2. The largest subview occupies the left-hand side and depicts the spectrum of the chosen well. The second subview occupies the bottom right-hand side. It appears only when an on/ off switch called “show adducts” is activated, Figures 2 and 3. It (15) McLafferty, F. W.; Turecˇek, F. Interpretation of Mass Spectra, Fourth ed; University Science Books: California, 1993; pp. 23-25.
a The adduct in italics is not incorporated in the quasi molecular ion discovery algorithm either in the Spectrum or 3-D map views.
Table 3. Average Stoichiometry of Hydrogen, Nitrogen, and Oxygen Relative to Carbon, in Five Diverse Novartis Combinatorial Chemistries chemistry
hydrogen/carbon ratio
nitrogen/carbon ratio
oxygen/carbon ratio
rack 165a rack 275 rack 508 rack 700 rack 998
1.61 1.23 1.11 0.96 1.29
0.22 0.12 0.12 0.12 0.15
0.11 0.27 0.09 0.09 0.13
a The A + 1 ratio of the N-substituted peptoid chemistry used to illustrate the 3-D map is comfortably accommodated by the use of the ratios from rack 165 in the ion current inclusion criteria 1c and 2c, Figure 1.
comprises a small discovery “scanner” box with the ∑ %RA as the y-axis, oval outlined in Figure 2. Under the x-axis, two buttons exist with arrows pointing right and left, respectively. The right and left arrows direct the quasi molecular ion discovery program to move up and down the spectrum, respectively, and scan for possible quasi molecular ions. Each time the respective arrow is mouse-clicked the discovery program skips to the next quasi molecular ion hit. As each ion hit is found, a histogram bar is displayed within the box with its height proportional to the ∑ %RA of the ions found in all the adduct positions. The last 20 consecutive hits are recorded within this box in blue. The current hit is displayed in red. The molecular weight and the ∑ %RA of the current hit are visible above the scanner box. In addition, when the show adducts switch is activated, an adduct stencil comprising dotted vertical lines appears on the large spectrum picture. When first switched on, the stencil [M + H]+ (or [M - H]- if in negative-ion mode) is positioned by default over the position of the expected quasi molecular ion. As soon as the quasi molecular ion discovery program starts scanning down or up mass upon user request in the discovery scanner box, the adduct stencil skips down or up the spectrum, respectively. 2.3. Quasi Molecular Ion Discovery in 3-D Ion Current Data Sets. The 3-D map of RackViewer13 presents a bird’s eye view of the entire ion current belonging to a 96-well rack using X axis, e.g., LC fraction no. or combinatorial synthesis well, Y axis (m/z), and color. Three colorization criteria are available, selectable with the list box color on the bottom right of the 3-D map Analytical Chemistry, Vol. 71, No. 24, December 15, 1999
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Figure 2. Positive-ion ESI spectrum of the expected synthetic product with monoisotopic molecular weight ) 566.3, in well E10, showing the prominent quasi molecular [M + H]+. As the spectrum has been zoomed in on the quasi molecular ion region, the gas dimers [2M + H]+, [2M + NH4]+, [2M + Na]+, and [2M + K]+ are off-scale. The quasi molecular ion discovery I algorithm found three molecular weight hits in this spectrum, i.e., 566.3 with ∑ %RA ) 258.6, 583.4 with ∑ %RA ) 29.9, and 1132.7 with ∑ %RA ) 110.8. The hit with mw ) 583.4, actually the [M + NH4]+ ion, is a false positive. The hit with mw ) 1132.7 is the [2M + H]+ ion. The discovery II algorithm found only the hits with mw ) 566.3 and 1132.7.
Figure 3. Diagram of information flow in the three 3-D map colorization schemes. The discovery algorithm detects all adduct ion current attributable to a quasi molecular ion and effectively relocates it to the quasi molecular ion m/z position. This ion current can then be rethresholded and color-visualized.
display, Figures 3 and 4. The criteria are by “Mass”, i.e., forbidden loss or adduct zoning, secondly by “Intensity”, i.e., simple %RA, and thirdly by “Discovery” (I and II), i.e., ∑ %RA. Upon entering the 3-D map, the default is by Mass rendering.13 This colorization is orientated towards an expected quasi molecular ion in each well’s spectrum13 and will not be discussed further in the present context. The colorizations that are of use for quasi molecular ion discovery are by Discovery (I and II) and to a lesser extent by Intensity. In both schemes each ion is colorized in a rainbow gradation according to its %RA, Figure 4, with red and blue 5560
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meaning high and low intensity, respectively. An illustrative vertical minimum/maximum color bar legend adjacent to 3-D map, appears upon colorization. In Discovery I mode, Figure 5, each putative quasi molecular ion displayed after colorization has to possess at least one adduct in Table 1 or 2 in the positive-ion or negative-ion mode, respectively, and this must fulfil the A + 1 relative intensity requirement already discussed. In Discovery II mode, Figure 6, each putative quasi molecular ion displayed after colorization has to possess at least two adducts in either Table 1 or 2, and this must fulfil the A + 1 relative intensity requirement. The Discovery I and II ∑ %RA current in the 3-D map is identical to that reported in the Discovery I and II scanner box in the Spectrum view. In both Discovery I and II, the colorization is then normalized versus the putative quasi molecular ion with the highest amount of attributable ion current, Figures 4 and 5. The attributable ion current is specifically ∑ %RA and has no isotope cluster breadth, i.e., for unknown monoisotopic cluster shapes, unlike the by Mass colorization in the 3-D map for expected monoisotopic cluster shapes. Using the discovery algorithm, all putative quasi molecular ions will be automatically displayed within this 3-D map of the total ion current. If too much obscuring ion current exists in any of the three 3-D map colorizations, then one can increase the threshold to eliminate it, which enhances the more important ion current, e.g., %RA ) 10% in the Intensity depiction or ∑ %RA ) 10% in the Discovery depiction. One can select a particular threshold with the list box “Threshold” on the bottom left of the 3-D map, Figures
Figure 4. 3-D map, colorized by ionization intensity (%RA). Each ion in every well is normalized to the base peak in each well, rather than rack-wide. The threshold is set to %RA ) 10%. Number of points ) 1625. For clarity, the yohimbine standard curve in row A is not displayed in the data in Figures 4, 5, and 6. Spectra (Figure 2) are directly accessible via a mouse click on the 3-D map points.13
Figure 5. 3-D map, colorized by the Discovery I program. The quasi molecular ions with the most attributable ion current are highlighted in red and orange. Each quasi molecular ion hit is normalized to the largest hit ∑ %RA in that well, rather than rack-wide. The threshold is set to ∑ %RA ) 10%. Nine putative quasi molecular ions have been oval-outlined, using the criterion of a contiguous track of at least three intense hits. From left to right the monoisotopic molecular weights are 349, 354, 539, 462, 405, 476, 566 (see Figure 2), 580, and 602, respectively. Number of points ) 339.
3 and 4. On first switching to the 3-D map, the threshold is defaulted to “dynamic” which rations displayable graphic points, well-by-well, rather than allowing noisy samples with many m/z values to grab a disproportionate share.
3. CONCLUSIONS The high-throughput and quick turnaround FIA-MS, designed to analyze the output from the Novartis corporate multiple parallel combinatorial chemistry program, generates yearly tens of thouAnalytical Chemistry, Vol. 71, No. 24, December 15, 1999
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Figure 6. 3-D map, colorized by the Discovery II program. The quasi molecular ions with the most attributable ion current are highlighted in red and orange. Each quasi molecular ion hit is normalized to the largest hit ∑ %RA in that well, rather than rack-wide. The threshold is set to ∑ %RA ) 10%. After requiring a second adduct to be present, four putative quasi molecular ions remain (oval-outlined) compared with Figure 5. From left to right the monoisotopic molecular weights are 539, 462, 566 (see Figure 2), and 580, respectively. Number of points ) 129.
sands of adduct-rich electrospray spectra. The choice of electrospray ionization was of critical significance and produces spectra that are well conditioned for automated estimates of spectral purities with reference to expected stoichiometric formulae. This soft ionization approach complicates any manual interpretation by the synthetic chemists, who are generally unfamiliar with the diversity of electrospray adduction patterns. However, this rich adduction proves to be optimal for automated molecular weight discovery as it allows searching for simultaneous adduct mass differences and then using these as pointers to putative quasi molecular ions. This is essentially a simple idea that has been latent since adducted quasi molecular ions, e.g., in methane positive on CI, first appeared in the mid 1960s. Its implementation required the availability of two 1990s technologies: a language for rapid development of visual interfaces, e.g., Visual Basic, and a robust and soft ionization technique that is compatible with liquid introduction, e.g., electrospray. The last deciding factor was the demand for highlighting tools to analyze industrial-scale FIA-MS
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data, whether from combinatorial syntheses, LC fractions, or natural products. The coincidence of these three factors enabled the development of a quasi molecular ion discovery software, both in a spectrum scanner view and in a 3-D map of the MS ion current. These quasi molecular ion discovery tools are built into the reporting system for our high-throughput FIA-MS, allowing their use by the Novartis synthetic chemists at their work benches. ACKNOWLEDGMENT Thanks go to Dr. Jan-Marcus Seifert, Department of Immunology, Novartis Research Institute GmbH, A-1235 Vienna, for providing the semi-preparative LC fractions for illustrating the quasi molecular ion discovery tools.
Received April 19, 1999. Accepted August 31, 1999. AC9904011
