Workshop Reports

Report on the Workshop on ‘High Mass Measurements in Mass Spectrometry’ Sponsored by the Committee on Quali- tative Organic Analysis, held at the 28th Annual Conference of the American Society for Mass Spectrometry, New York City, New York, May 1980

Various methods of mass analysis were presented for consideration: (1) high field magnets-adapted to com- mercial mass spectrometer systems, (2) large radius magnet systems, ( 3 ) low frequency quadrupole mass analyzers, and (4) time-of-flight analyzers (such as used with ’”Cf desorption). Other methods were proposed by attendees. For example, William Aberth suggested a Wien filter which has a demonstrated resolution of 3000 and an upper mass limit which is yet to be determined. Robert McIver pointed out that an ion cyclotron resonance spectrometer equipped with a superconducting magnet a t 120 kilogauss may produce unit resolution at m / z 10000. Upper mass limits of at least m / z 1000 have been demonstrated at about 20 k G using Fourier transform techniques.

Commercial magnet systems were described by Roy McDowell of Imperial College, London. He pointed out that electromagnets equicped with carbon- steel pole faces can be used to study masses up to about m / z 1000 at 8 kV of acceleration (as on a Kratos MS 50, for example). ‘Extended field’ magnets have been introduced. They are equipped with silicon-steel pole faces and can be used up to m / z 1750 at 8 kV. Finally, cobalt-steel pole faces have been used on ‘high field’ magnets (up to m / z 3000 at 8 kV). The ‘extended’ and ‘high field’ magnets cannot be scanned such that equal time is spent on each m / z signal. In fact, more time is spent on the higher mass peaks, and this may be an advan- tage.

Fred McLafferty reported on a tandem mass spectrometer that he and his co-workers have constructed at Cornell University. Ultimately the instrument will be equipped with two large radius, high field magnets which should permit mass selection, activation, and mass analysis of ions of mass greater than 3000. This instrument may be use- ful for analysis of mixtures containing high molecular weight components (such as complex oligopeptides).

A low frequency quadrupole was described by Lewis Friedman. H e poin- ted out that an upper mass of 100 000 can be obtained by reducing the

frequency applied to the rods by a factor of 10. This increase of a factor of 100 in mass limit is because the mass is inversely proportional to square of the frequency. In fact masses in the range of 8 0 000 have been investigated using a system located at Brookhaven National Laboratory. Dr Friedman pointed out that correctly designed rods and power supplies may give a resolving power of 20 000.

A high mass instrument has been constructed at LaTrobe University using a 78 cm magnetic sector and a 100 cm electrostatic analyzer. Using this instrument, polypropylene glycols with nominal mass-average relative molecu- lar masses of 1000,2000,3000 and 4000 have been studied using field desorption. The highest mass ion observed to date is m / z 7400.

Methods of vaporization and ion- ization were also considered. Various techniques were proposed: (1) field desorption, (2) rapid heating, (3) fission fragment desorption and (4) electro- spray techniques. Gordon Wood from the University of Windsor has used conventional mass analysis to charac- terize polyethylene glycol oligomers by forming multicharged ions by attach- ment of one or more [Ba]” ions to the polymer component molecules.

The results of a detailed analysis of various polystyrene fractions were dis- cussed by Robert Lattimer of B. F. Goodrich. His work was conducted on the ‘high field’ MS 50 at the Johns Hop- kins NSF Regional Instrumentation facility. Average molecular weights up to about 5400 were determined using field desorption and integrating scans. The results were in good agreement with those determined using vapor phase osmometry.

Although field desorption appears to be a useful vaporization/ionization method for molecular weights in the 1000 to 3000 range (and higher for synthetic polymers), the technique does not appear applicable to higher molecu- lar weight materials, particularly bio- polymers. This was demonstrated in a 1976 study of glucagon, a peptide containing 29 amino acids, by Winkler, Beuhler and Friedman. They employed a single sector magnetic instrument with a 152 cm radius and field desorption ionization. The highest mass signal observed was m / z 2110 which has 18 amino acids. D r Friedman pointed out that the rapid heating procedures will have a similar upper mass limit. Both techniques suffer from the relatively

large desorption energy of biomolecules which, for a molecule containing many polar groups, will exceed bond energies.

A method that appears promising for high molecular weight materials is the electrospray technique developed by Malcolm Dole. Both Professor McLafferty and Dr Friedman discussed aspects of this technique, which involves rapid evaporation of tiny droplets which are formed by electrospraying solutions into the source. Each droplet contains ideally one molecule of analyte. Profes- sor McLafferty has proposed to couple a high pressure liquid chromatograph and his tandem MS/MS instrument for stu- dies of high molecular weight substances. He speculated that col- lisional activation may be important for removing the last few molecules of solvent.

Jerry Hunt, a co-worker of Professor R. D. McFarlane at Texas A and M University, reported on recent experi- ments using an alternative method of ionization: i.e. with 252Cf fission frag- ments. Synthetic nucleotides up to mass 12 000 have been studied; only [M + HI’ ions were observed in the positive ion mode, whereas extensive fragmentation yielding sequence information was found for negative ions. Furthermore, chlorophyll clusters have been seen up to m / z 8000, and these may be useful as mass standards. A sample size of about 100 micrograms is required, and 5 to 6 hours are required to complete the experiment. A discussion ensued on whether post-acceleration is necessary for triggering an electron multiplier with high mass particles.

Another issue considered briefly was mass calibration. This may not be a serious problem; fomblin can be used in the 1000-3000 amu range. Dr Friedman suggested that calibration will be no problem at higher masses either. One needs only to expand into the source rather common solvents using a jet to grow clusters which have an established repeating unit.

An interesting discussion occurred on whether mass spectroscopists should tackle the problem ’of high molecular weight materials or defer to more clas- sical technologies such as ultracentri- fugation or light scattering. There are compelling reasons to pursue high mass measurements in addition to the scien- tist’s quest to constantly improvc Ili\

methodology. For example. c!.i+.it a , techniques can never yield exact mole- cular masses. Furthermore, mass spectrometers produce information on

0 Heyden & Son Ltd, 1980 BIOMEDICAL MASS SPECTROMETRY, VOL. 7, NO. 8, 1980 365

WORKSHOP REPORTS

molecular weight distributions for mix- tures and on the general purity of the sample, provided fragmentation can be minimized in the vaporization and ion- ization steps. Finally, as pointed out by Dr Friedman, the development of controlled nuclear fusion may depend

entirely on our capability for high mass The author is grateful to Drs P. G. measurements. Space charge effects Kistemaker and R. J. Cotter for assis- currently limit current density in fusion plasmas, and this can be minimized with ion clusters. Of course, the study of large ion clusters will require high mass MICHAEL GROSS measurements. University of Nebraska

tance in preparing this report.

Report on the Workshop on ‘Quan- titation by Positive or Negative Chem- ical Ionization’ Sponsored by the Committees for Forensic Applications and for Quantitative Analysis, held at the 28th Annual Conference of the American Society for Mass Spectro- metry, New York City, New York, May 1980

This workshop was held using a panel of experienced chemical ionization (CI) users to answer questions posed by attendees. Sandy Markey of NIH was the Chairman and the panelists were A1 Yergey of NIH, Bill Garland of Hoffman-LaRcche, Dave Hachey of Argonne, Dwight Matthews of Washington University, Fred Falkner of Pfizer and Rodger Foltz of the Uni- versity of Utah. All panelists described their applications which centered upon quantitation of compounds of bio- chemical or pharmaceutical interest. Most used gas chromatography mass spectrometry (GCMS) although several applications did utilize direct probe sample introduction. Following these introductory remarks, questions of quantitation by CI came from the floor.

A question was asked about the linearity and precision of CI compared with electron impact (EI) methods. Dwight Matthews said that they were similar, but that the CI assays were cleaner. This seemed to be agreed on, but later a series of questions about factors leading to variability were dis- cussed. It was agreed that source temperature is a very critical parameter, perhaps more so in negative CI (NCI) assays. In most assays the source temperature should be carefully stabil- ized, if not actually controlled. There

were no special problems noted with the use of a fragment ion rather than a pseudomolecular ion in CI quantitative analysis.

There was agreement about the pro- cedure for optimizing and Ieproducing the CI conditions. The source pressure should be adjusted to maximize the signal from the analyte, and then the characteristics of the reagent gas (intensity, ratio of primary, secondary, tertiary, etc. ions) noted. Subsequent analyses would begin by re-establishing that set of reagent gas parameters. Favorite tune-up compounds for NCI were given as fomblin with nitrogen or methane as described by Adries Bruins in a poster session. Other compounds such as perfluorotributylamine give spectra useful in only a very limited mass range (400-600).

There was a discussion of how much improvement in sensitivity could be expected with NCI as compared with EI. It was noted that such a comparison must be properly made; that is, the best E I conditions versus the best CI conditions. It is unlikely that both these would result from the same source conditions. Sandy Markey said his NCI biogenic amine assays were approximately 30-fold more sensitive than the best El he could do. In addition to the possible increase in absolute sensitivity which the cascade of low energy electrons in the source produces for NCI, the selectivity of the ionization process generally resulted in cleaner backgrounds, thus giving an increase in abundance sensitivity. Asked whether saturation in NCI was a prob- lem, the panelists felt that at the low levels normally used, less than 100 ng, this was not a problem, especially when using natively capturing compounds.

When derivatization is used, relatively large amounts of endogenous materials may capture the bulk of electrons and reduce sensitivity, as well as give rise to very large interfering background ions, similar to those encountered in EI or positive CI (PCI) of derivatized extracts.

There were several questions about reagent gases. Why is methane the best moderator in electron capture NCI? It does work better than nitrogen, helium or argon, presumably because its vibra- tional energy states moderate the mean electron energy to lower values than the other simpler gases. Isobutane was said to work equally well. Both are good third-body stabilizers. Ammonia was suggested for PCI assay of saturated hydrocarbons, while water was sugges- ted for olefins. Although not as widely applicable, ammonia produces the cleanest PCI spectra. Otherwise, iso- butane is recommended.

It was noted by a member of the audience that all the panelists have quadrupole instruments of one design or another. Did that mean that they were inherently better for CI? The answer was no, but their low voltage operation meant that the electrical discharge problems were reduced compared with magnetic machines. It was also noted thrt quadrupoles appear to tolerate elevated analyzer pressures better than magnetic instruments. On the other hand, quadrupoles seemed much more susceptible to contamination in the souce and multiplier regions. There was no consensus on whether isobutane caused more contamination than either methane or ammonia.

FRED ABRAMSON George Washington University

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