calibration point for liquid secondary ion massspectrometry tandem mass spectra measuredwith an ebqq...

JOURNAL OF MASS SPECTROMETRY, VOL. 31, 280-288 (1996)

Calibration Point for Liquid Secondary Ion Mass Spectrometry Tandem Mass Spectra Measured with an EBqQ Hybrid Mass Spectrometer

Sharon W. Lemire and Kenneth L. Busch* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA

A protocol for establishing standard instrument conditions for the measurement of product ion tandem mass spectra from parent ions generated by liquid secondary ion mass spectrometry (LSIMS) is presented. The ion at m/z 319 (C') from 2-butyltriphenylphosphonium bromide is selected as the parent ion and then is subjected to collision-induced dissociation. The relative abundances of product ions at m/z 263 and 183 are monitored as a function of collision energy while keeping the target gas pressure constant. The collision energy at which these two most abundant product ions are equal is defined as the calibration point at that collision gas pressure. LSIMS tandem mass spectra acquired at the calibration point show good reproducibility for several model compounds. Spectra obtained at the calibration point at two different collision gas pressures, representing multiple and single collision conditions, also show a high degree of similarity. Use of this protocol may aid in the establishment of a tandem mass spectral library.

KEYWORDS : calibration; MS/MS; fast atom bombardment; liquid secondary ion mass spectrometry; library spectra; collision-induced dissociation; tandem mass spectromety

INTRODUCTION

The collision-induced dissociation process that forms the basis of tandem mass spectrometry (MS/MS) can be carried out on instruments with a geometry represented by XCrflQ, [X =BE, EB, EBE or Q and [rf] = q(uadrupole), h(exapole), or ~(ctapole)]. In each case, the low collision energy regime used means that the reproducibility of product ion spectra is dependent on instrumental parameters such as type and pressure of collision gas, collision energy, and rf tuning. These parameters are difficult to reproduce from instrument- to-instrument, or even from day-to-day on the same instrument. For comparison of product ion MS/MS spectra obtained on different instruments, or to develop a library-quality database of such spectra, instrument- independent spectra must be reproducibly acquired. Generation of reproducible MS/MS spectra requires a calibration method that is simple, reliable, applicable to a wide range of compounds, and independent of the type of collision gas.

Dawson and Sun' proposed a calibration method for electron impact (EI) MS/MS in which the collision pressure (target thickness) and collision energy are kept constant. However, a known and reproducible target thickness is difficult to achieve on different instruments

* Author to whom correspondence should be addressed.

solely by control of these parameters, since the location of the gauge used to read the pressure of the collision gas varies with different manufacturers. Other instrument design variations, such as quadrupole rod radius, can also make it difficult to achieve a reproducible collision condition. The relatively scattered results of a round-robin test utilizing the above-mentioned method were the motivation behind the idea that standard operating conditions be based on a suitable ratio of product ions in a tandem mass spectrum. The ideal calibration compound would have at least two product ions for which the abundance ratio is largely dependent upon collision energy, and two product ions for which the abundance ratio is largely dependent upon target thickness. This 'Holy Grail' of MS/MS has yet to be located.

Recently, Naylor and Lamb' suggested a calibration method applicable to both EI MS/MS and fast atom bombardment/liquid secondary ion mass spectrometry (FAB/LSIMS) MS/MS that utilized the nodal properties of an r.f.-only quadrupole. Since the energy at which nodes occur is mass dependent rather than compound- dependent, a collision energy may be selected based on the energy at which a node occurs in the energy- resolved tandem mass spectrum of a calibration compound. The calibration compound is used to identify these nodes and to reach a set of standard operating conditions. In general, the calibration compound should produce an abundant ion with mass similar to the analyte parent ion. This requirement precludes the use of a 'universal' calibrant applicable to a range of compounds of different molecular masses. In addition, ion

CCC 1076--5 174/96/030280-09 0 1996 by John Wiley & Sons, Ltd.

Received 8 September 1995 Accepted 14 November 1995

281 CALIBRATION OF LSIMS MS/MS O N A HYBRID INSTRUMENT

transmission nodes are pronounced in quadrupole collision cells, but are progressively damped in hexapole and octapole collision cells, limiting the usefulness of this effect as well.

Martinez3 presented an EI-MS/MS calibration method applicable to all multipole collision cells. The compound selected for calibration, acetone, is not amenable to FAB/LSIMS ionization, however. Martin- ez’s approach would require setting up the instrument in the EI mode for calibration, and then switching to the FAB/LSIMS mode for analysis.

The calibration method presented here uses an empirically determined calibration point to acquire reproducible product ion tandem mass spectra from parent ions generated by LSIMS. The calibration point is based on the measurement of equal abundances for the two most abundant product ions from a calibration compound. At any set target thickness, the collision energy is increased until this condition is met. Specifying the requirement of equal ion abundances increases the flexibility of the experiment by eliminating the need to attain a known and reproducible target thickness. The collision energy selected for acquisition of analyte product ion spectra is simply the energy at which the relative abundances of the two most abundant product ions of the calibration compound are equal at a constant pressure. A similar protocol for EI- MS/MS has recently been de~cribed.~ That procedure used a calibration compound for which parent ions were generated by EI ionization; our goal was to use a compound that produced an intense, high-quality LSIMS or FAB mass spectrum.

EXPERIMENTAL

The calibration point method described in a previous paper4 for the acquisition of reproducible EI-MS/MS product ion spectra was modified to record product ion tandem mass spectra for compounds ionized by LSIMS. A VG7O-SEQ (VG, Manchester, UK) hybrid mass spectrometer of EBqQ geometry (q is the collision quadrupole and Q is the final mass-analyzing quadrupole) was tuned to a standard set of MS/MS conditions as described below. Product ion tandem mass spectra were acquired in the multi-channel analyzer (MCA) mode under these standard conditions. The primary ion beam, produced by a thermionic cesium ion gun operated at 35 kV, was used to sputter positive secondary ions from the sample solution. The instrument was operated at a mass resolution of 1300 (baseline definition). Product ions were produced by collision-induced dissociation (CID) in an r.f.-only quadrupole collision cell with argon as the collision gas and the r.f. amplitude set at 75 V. The analyzing quadrupole was operated at unit mass resolution.

Multiple collision (MC) conditions were obtained by introducing argon into the collision cell until the parent ion (m/z 393 for CsI) was attenuated by - 80%, producing a reading on the ion gauge nearest the collision cell of 2.3 x lo-’ mbar. Single collision (SC) conditions were achieved by attenuating the same parent ion by -25%, producing an ion gauge reading of 5.4 x

mbar. These readings have been corrected for argon response. The actual collision gas pressure within the cell is unknown, and is estimated as a few mbar.5s6 One of the advantages of the method proposed here is that accurate measurement of the collision gas pressure is not required since the calibration point is empirically defined as the collision energy at which the two most abundant product ions of the calibration compound have equal abundances at a constant gas pressure.

The instrument was tuned using m/z 393 from solid (CsI)Cs+ to produce a well resolved parent ion signal. The argon collision gas was then introduced into the collision cell until the parent ion was attenuated by the desired amount. Tuning parameters were re-checked for the parent ion of the calibration compound at the indi- cated collision gas pressure, The collision energy was then increased to the first collision energy at which the abundances of the two most abundant product ions of the calibration compound appeared to be approximately equal. The tandem mass spectrum of the calibration compound was then acquired, and ten scans were added together to establish the abundances of these two ions. The collision energy was fine tuned in 1 eV increments until the abundances of the two ions were as nearly equal as possible. This energy was defined as the calibration point, and all other tandem mass spectra were acquired under these conditions. The calibration point (in eV) was located on a day-by-day basis prior to the analysis of the model compounds.

Model compounds were chosen to represent different classes of compounds commonly analyzed by FAB or LSIMS. Evaluated compounds (and the corresponding abbreviations used in this paper) were neat m- nitrobenzyl alcohol (m-NBA, M , 153), harmine methio- dide (HMI, C+ 227), thiamine hydrochloride (vitamin B,, C+ 265), diphenylthiophene sulfonium phosphorus hexafluoride (SULF, C+ 269), n-butyltriphenylphosphonium bromide (n-BTPP, C + 319), acridine orange-10 nonyl bromide (A-1372, C + 392), diphenyl 4- dodecoxyfluorophenylsulfonium antimony hexafluoride (New Sulf, C+ 465), l,l’-didodecyl-3,3,3’,3’-tetra- methylindocarbocyanine perchlorate (D-383, C + 666) and 3,3’-dihexadecyloxacarbocyanine perchlorate (D- 1125, C+ 726). These compounds covered a mass range from 153 to 726 Da, allowing a study of the useful mass range of the calibration technique. The model compounds were used as obtained from commercial sources; sample/matrix solutions of N 0.05 g of sample per gram of m-NBA were prepared. Approximately 1 pl of each solution was deposited on the probe tip. LSIMS mass spectra of all the analytes were acquired over several days under both MC and SC conditions.

The matrix m-NBA was chosen because it provides long ion lifetimes in the ion source and because it readily solubilizes the calibration compound and the model compounds. Strong, stable signals from the calibration compound were also observed in the following matrices: 4-hydroxybenzenesulfonic acid, nitrophenyl octyl ether, glycerol, thioglycerol and 2-hydroxyethyl disulfide. The calibration point was reached for the calibration compound in each of these matrices, but at different collision energies. Preliminary data, stemming from this matrix study, suggest that the calibration point may be reached at lower collision energies when

282 S. W. LEMIRE AND K. L. BUSCH

glycerol is used as the matrix. This phenomenon is being further investigated; all of the data discussed here were obtained with m-NBA.

RESULTS A N D DISCUSSION

The salt 2-butyltriphenylphosphonium bromide (2- BTPP) was chosen as the calibration compound because it is easy to handle and produces a stable, long- lived signal in positive-ion LSIMS. The intact cation of this compound at m/z 319 was selected as the parent ion and subjected to CID. Product ions at m/z 263 and 183, produced by losses of C4H8 and CI0Hl6, respectively, are the two most abundant product ions in the product ion tandem mass spectrum. The relative abundances of these three ions were monitored as a function of collision energy at two pressures representing MC and SC conditions. Under MC conditions, the collision energy at which the abundances of the two product ions at m/z 263 and 183 were equal ranged from 29 to 49 eV with a day-to-day reproducibility of f9 eV, and from 162 to 185 eV with a day-to-day reproducibility of f 10 eV for SC conditions. The collision energy variations represent pressure variations rather than an inability to control the collision energy electronically. The calibration point is taken as the first collision energy at which the abundances of the two product ions at m/z 263 and 183 are nearly equal. On most days, the abundances of the two ions were within 5% of each other. However, at the calibration point, a relatively small change in collision energy (2-3 eV) can result in a 5-10% change in the relative intensity difference between these two ions, so that a total difference of 10-15% may be considered as acceptable. Under the conditions represented in Fig. 1, the calibration point was reached at 49 eV (the cross- point of ion abundances). The collision energy at which the relative abundances of these two ions are nearly equal remains constant to within _+2 eV throughout the analysis as long as the gas pressure in the collision cell is allowed to stabilize prior to the analysis. The collision energy in the laboratory frame of reference is given by the instrument software as the nominal difference

Y I , > . 4

30 40 50 Cdlisim Energy (ev)

/ T I 9 + Xi- lq Figure 1. Plot of the relative abundances of the parent ion at m/z 31 9 and the product ions at m/z 263 and 183 of 2-BTPP versus the collision energy, showing a typical calibration point. The collision gas was air at a pressure of 2.2 X lo-’ mbar and the collision energy was ramped from 20 to 60 eV at 2 steps per scan.

between the ion source potential and the d.c. level of the quadrupole collision cell.

The tandem mass spectra of the nine model compounds, selected from compounds known to run well in LSIMS, were statistically assessed. For each tandem mass spectrum, the seven most abundant ions in the spectrum were chosen for abundance tabulations and statistical analyses. Calculations included mean relative abundances, standard deviations and similarity indices. Since the primary difference between spectra obtained from day to day is the relative abundances of the product ions with respect to the parent ion rather than the relative abundances of the product ions with respect to each other, the relative abundances of the product ions were renormalized to the most abundant product ion before statistical calculations were performed. Product ions of low relative abundance ( < 2%) were excluded from the final statistical evaluation owing to their large relative deviations and their low signal levels. When it was necessary to choose between ions of similar abundance, the higher mass product ions were chosen over the more common lower mass ions.

A similarity index, calculated according to Eqn (l), expresses the relative percentage difference between the abundances (I) of two ions of the same mass:

{ I - I b + zb)/2) loo% (1) where the subscripts, a and b, refer to the relative abundance of these two ions in two separate runs. Each model compound was analyzed four times under both MC and SC conditions. To evaluate the run-to-run reproducibility of ion abundance, the similarity index for each ion was calculated between each run, giving a total of six comparisons that were then averaged to yield a mean similarity index for each ion. A mean similarity index was calculated for each ion under both sets of collision conditions. Assessment of the overall spectral similarity for each model compound was based on the mean similarity index averaged over all the ions evaluated.

Table 1 contains values for the means, the standard deviations and the mean similarity indices under both collision conditions for the seven most abundant ions of all the compounds studied here. The standard deviation of the mean relative abundance, averaged over four runs, was calculated for each ion under both sets of conditions. The standard deviation most accurately represents the run-to-run spectral reproducibility. Relative comparisons, such as the similarity index (imposed on previous data4), create the illusion of poor reproducibility when low product ion abundances are involved. Visual comparisons are the easiest, but are, of course, not numerically treatable. Figure 2 compares the variation of ion abundances from run-to-run under MC conditions for three model compounds (m-NBA, n-BTPP and D-1125). Together, these compounds encompass the entire mass range studied in this work. The lowest mass compound, m-NBA, appears to have the least overall variability under MC conditions (as assessed by the closeness of the points shown on the graph). However, Table 1 indicates that n-BTPP, based on an overall similarity index of 15%, has the least statistical variability. The larger number of lower abundance ions in the product ion tandem mass spectrum of

CALIBRATION OF LSIMS MS/MS ON A HYBRID INSTRUMENT 283

Table 1. Results of the statistical analysis of the seven most abundant product ions

Compound

m-NBA

Overall similarity

HMI

Overall similarity

Vitamin 8,

Overall similarity

Sulf

Overall similarity

n-BTPP

Overall similarity

A-1 372

Overall similarity

New sulf

Overall similarity

D-383

Overall similarity

mlz

63 77 89 95

107 124 136

115 143 158 169 184 197 21 2

81 122 144 177 182 207 224

77 115 128 147 154 160 192

108 183 185 199 262 263 275

222 223 236 250 264 265 266

77 107 141 170 220 388 465

31 2 336 352 481 494 636 650

Mean +standard deviation M C

10*3 23 * 2

l o o t 0 7 *1 9 + 1 5 * 2 2 *l

3 * 1 2 *1 6 *3

15*10 89 *23

2 *l 77 *20

22 1 4 100 *o 26 * 3

0.3 0.2 1 *0.1 1 *0.4

0.4 * 0.3

6 +2 3 9 * l l 40*3 12+3 7 * 2

84*19 84*18

50 * 9 l O O * O 34 +6 4 * 1

1 9 * 2 23 * 2 11 +1

11 *1 24 *5 11 *1 55 * 9 11 * 4 51 +5

100 *o

1 t 0 . 5 3 *0.3 1 *I

100*0 12+1

0.3 * 0.2 4 * 3

4 9 + l l 91 *15 14*1 11 1 5 8 + 1

53 +50 47 *49

sc

9 *3 55*7

l O O * O 7 * 1

47 * 8 50*18 1 6 * 5

6 + 2 3 * 1 4 * 2

37*16 50*16 10*3

100 + o

17*3 100*0 20 * 4

1 +0.3 1 k0.5 2 * 1 1 *1

28*14 28*13 13*5 12 * 4 4 t l

32 *6 100+0

43*15

45+12 37 * l o 51 * 6 22*4 29 A4

l o o t 0

14*3 17 * 4 10*4 70*12 41 *11 91 *16 91 *14

6 * 2 4 * 2 1 +1

10010 27 * 6

2 * 1 66+11

33 f 24 55 f 26 13*9 15*9 16*18 58 * 29 98*5

M C

34 9 0 8

13 51 44 23

19 44 63 90 30 52 34 47

25 0

16 54 28 41 96 37

36 36 42 33 34 27 26 33

23 0

19 21 14 11 16 15

9 26 11 21 45 12 0

18

38 12 88 0

12 84

118 50

29 19 12 54 14

130 144 57

Mean similarily index sc

32 15 0 8

22 48 43 24

40 19 67 54 37 35 0

36

22 0

27 45 83 65 89 47

66 60 49 40 34 24 0

39

41 0

34 36 15 23 18 24

26 30 47 22 32 20 18 28

36 43 57 0

26 33 19 31

82 59 89 81

100 54

5 67

MC M. SC

8 84 0 0

135 164 186 79

49 56 26 87 56

135 26 62

25 0

26 84 34 75

123 52

126 33

102 6

49 90 17 60

15 0

27 161 91

4 87 55

24 32

5 25

117 57 10 39

127 43

7 0

75 146 179 82

39 49

7 31 63

9 71 38


Table 1. (Continued)

Compound mlz

D-1125 159 196 259 277 342 368 501

Overall similarity Average difference percentage

Mean *standard deviation MC sc

1 1 *10 27*20 16*11 14*11 10 * 6 29 20 68*11 80*24 75 *50 79 *29 48*13 47*28 26 * 6 31 * 8

Mean similarity index MC SC MCvs.SC

93 88 85 84 85 9 98 88 98 20 40 17 0 49 5

34 58 1 28 34 19 51 63 33 37 40 56

m-NBA, as pointed out above, skews the statistical results. We have not, as yet, implemented a mass- dependent weighting factor that would attenuate the significance given to lower mass ions and possibly produce statistical results more in accord with the visual assessment. The highest mass compound, D-1125, with an overall similarity index of 51%, shows the greatest spectral variability among these three compounds. In general, spectral reproducibility for both MC and SC conditions, as shown on the primary axis of Fig. 3, deteriorates as the masses of the model com-

" 0

$ 4 I

63 77 89 95 107 124 136

n-BTPP 8

183 185 199 262 263 275

120 1- I

I Dl125

1 B f

159 1% 259 277 342 368 501 mlZ

7 I Run1 Run2 Run3 X Run4

I

Figure 2. Plot of the relative abundances of the product ions of rn-NBA (top), n-BTPP (center) and D-1125 (bottom) showing the variation in abundance over four runs under multiple collision conditions.

pounds deviate in either direction from the mass of the calibration compound (2-BTPP), with the obvious exception of m-NBA. For MC conditions, the overall spectral similarity ranged from 15% for n-BTPP to 57% for D-383, with an average difference of 37%. The reproducibility under SC conditions, although not as good as for MC conditions, ranged from 24% for n-BTPP to 67% for D-383, with an average difference of 40%. The average difference percentage, defined as the overall spectral similarity for spectra acquired under the same conditions averaged over all the compounds evaluated, is also given in Table 1.

A possible explanation for the observed deterioration in reproducibility as the masses of the model compounds deviate from the mass of the calibration compound involves the nodal properties of quadrupoles. Ion motion through an r.f.-only quadrupole may be described as sinusoidal about the longitudinal (z) axis of the quadrupole collision cell with a peak-to-peak amplitude greater than the dimensions of the exit aperture. Maximum ion transmission occurs for ions that enter and exit the collision cell close to the z-axis. This condition is met when the wavelength of the ion trajectory is such that the ions traverse an integral number of half- wavelengths between the entrance and exit apertures. Conversely, a transmission minimum is observed when the ion traverses an half-integral number of half- wavelengths over this distance so that the ions are off- axis at the aperture. A plot of ion abundance versus collision energy shows a series of transmission maxima and minima in response to these two extreme cases. As the slope of this plot approaches zero near a maximum or a minimum, the relative abundances in this region of the curve vary less with collision energy than those in steeper regions of the curve. Under a standard set of collision conditions that result in a transmission maximum for the parent ion of the calibration compound, model compounds having ion trajectories that result in a maximum or a minimum under these standard collision conditions should have better reproducibility than model compounds having ion trajectories that do not result in such maxima or minima. The latter compounds would have maxima or minima at collision energies in the steeper region of the curve on either side of the calibration maximum, resulting in worse reproducibility. The following discussion presents a relative method for quantitatively correlating the observed pattern of reproducibility


S o -

n /

O---c-‘t- 1 - x -* e 154 227 266 26s 319 592 46S 666 726

m/Z

-t Similarity Index --t Ab.dutoDevi8tbn

Figure 3. For multiple (top) and for single (bottom) collision conditions, the similarity index for each model compound is plotted versus m/z and is shown as the primary axis on the left. The absolute deviation of the ratio (rnr/m2)”* from the calibration maximum is plotted versusrn/z for each calibration compound and is shown as the secondary axis on the right.

among the model compounds evaluated here to the nodal properties of the quadrupole collision cell.

The wavelength of the sinusoidal trajectory of an ion is dependent on both collision energy and ion mass according to Eqn (2) taken from Alexander et aE. :7

A = 4(Elab)1/2/f4m”2 (2)

4 = 4 evr.f./Cm(2.fr)21 (3)

where q is expressed by7

Elab is the axial kinetic energy of an ion as determined by the collision energy in the laboratory frame of reference, q is the Mathieu parameter [Eqn (311 that describes ion motion through an r.f.-only quadrupole, f and q.f. are the r.f. frequency and amplitude, respectively, of the collision cell, r is the radius of the inscribed circle of the quadrupole and rn and e are the mass and the charge, respectively, of the ion. The number of half- wavelengths traversed by an ion between the entrance and exit apertures of a quadrupole of length L can be expressed by Eqn (4)7 in terms of the number of nodes, n :

n = L/(A/2) (4)

Transmission maxima occur when n assumes integral values and minima occur when n assumes half-integral values. In the calibration point experiments described here, the adjustable parameters in the above equations were optimized for the mass of the calibration compound so that we may assume that n is an integer and we are at or near a transmission maximum. The ratio of the mass of the calibration compound to the mass of any of the model compounds may be used to determine the number of nodes traversed by the model compound. The ratio of n2 for a model compound to n , for the

calibration compound is, according to Eqn (4), n,/nl = Al/A,. By substituting Eqn (3) for q into Eqn (2), it can be seen that the ratio & / A z = (rnl/rn2)’~2 so that n2 = n,(rn,/rn2)1/2. Assuming that under our standard collision conditions n , is an integer, then when (rnl/rnz)’/2 assumes integral values, n, will also be an integer and the model compound will traverse an integral number of nodes corresponding to a maximum in ion transmission for that model compound. On the other hand, when this ratio assumes half-integral values, n2 will only have integral values if n1 is an even integer; n, will have half-integral values when n, is an odd integer and the model compound will traverse a half-integral number of nodes corresponding to a minimum in ion transmission for that model compound. If n2 of the model compound assumes integral values, then the standard set of collision conditions are such that one of the maxima for that model compound corresponds to the maximum for the calibration compound. Conversely, if n2 assumes half- integral values, one of the minima for that model compound corresponds to the calibration maximum. The difference between the value of (rn,/rn2)1’2 and the nearest integer or half-integer represents the expected absolute deviation of the calculated transmission maxima or minima for each model compound from the calibration maximum. Model compounds with the lowest absolute deviation from the calibration maximum should have the best reproducibility. The reproducibility should decrease as the absolute deviation increases. Figure 3 plots the absolute deviation of (m,/m,)112 from the calibration maximum on the secondary y-axis and the similarity index for multiple collision conditions on the primary y-axis versus m/z. The similarity in the shapes of these two curves based on different mathematical calculations is striking. A similar plot for single collision conditions, also shown in Fig. 3,


follows the same general trend. These results confirm the expected effects of quadrupole transmission maxima on ion reproducibility and independently validate our experimental approach and similarity index calculations as directly reflective of operating parameters for quadrupole collision cells.

Equation (1) was also used to calculate a mean similarity index between product ion tandem mass spectra acquired under MC conditions and those acquired under SC conditions for each model compound. In this case, I, and I, would refer to the mean relative abundance of the specified ion under MC and under SC conditions, respectively. Table 1 shows the similarity of product ion tandem mass spectra measured under the two pressure regimes in terms of the mean similarity index (designated MC us. SC) for all the model compounds. In general, the degree of similarity between MC and SC conditions increases with mass. This result is probably a consequence of the fact that more energy is generally required to produce fragmentation in higher mass compounds. Since the extent of fragmentation changes with collision energy, a calibration point energy based on a low-mass calibration compound may not be sufficient to allow parent ion fragmentation by pathways other than those requiring the minimum amount of energy. Assuming that only the minimum energy condition was satisfied under both MC and SC conditions, the spectra acquired under these two conditions, as represented by the mean relative abundances, should be most similar for the highest mass compound, D-1125, as is seen in Table 1. Since n-BTPP is isobaric with 2-BTPP, the calibration point energy should be sufficient to produce spectra with a similar degree of fragmentation under all collision conditions, as is also reflected in the data shown. The lowest mass compound, m-NBA, is most affected by differences in collision conditions, as expected, because the amount of energy deposited under calibration point conditions is likely to exceed by far the minimum energy needed for the dissociation. The amount of excess energy varies

89

1

with collision conditions, and fragmentation may occur by several different pathways, increasing the differences between the spectra. Comparisons of Figs 4-6, showing the tandem mass spectra for m-NBA, n-BTPP and D-1125 acquired under MC conditions (top) and SC conditions (bottom), support these observations. The calculated overall spectral similarity between the two separate pressure regimes ranged from 33% for D-1125 to 82% for New Sulf, with an average difference of 56%. These results indicate that, with the proper choice of calibration compound, instruments incapable of reach- ing collision energies greater than 50-100 eV should be able to acquire reproducible product ion tandem mass spectra under MC conditions by increasing the target gas pressure until the calibration point can be reached at lower collision energies. Furthermore, the spectra obtained on these low collision energy instruments should be comparable to spectra obtained on other instruments under different collision conditions.

The fundamental question of this study is whether a reproducible set of conditions can be found for LSIMS MS/MS such that spectra are similar when obtained on different instruments. In absolute terms, compound identification will rely on measurement of the tandem mass spectra of the unknown and the standard compound sequentially on one instrument. In real terms, sample identification relies on the comparative evaluation of the pattern of ion masses and their abundances. The direct correlation of ion masses, on a one-by-one basis, is assumed. Both standard and unknown must produce a product ion of the specified mass. The work here deals with the acceptable variations in ion abundance values. Variations in product ion abundances are propagated by the inherent variabilities in abundances for parent ions generated by LSIMS. At lower concen- trations, one may expect even greater variability in ion abundances than those encountered here, where a relatively high analyte concentration was used. The relationship between absolute signal intensity, standard deviation and the number of scans required to reduce

mlz

Figure 4. Comparison of the tandem mass spectrum of m-NBA acquired under multiple collision conditions (top) with the tandem mass spectrum acquired under single collision conditions (bottom), Both spectra were acquired at the calibration point for 2-BTPP. Deviation from Table 1 - 79%.


1007

183 n-Butyltriphenylphosphonium

Cation i 163

Figure 5. Comparison of the tandem mass spectrum of n-BTPP acquired under multiple collision conditions (top) with the tandem mass spectrum acquired under single collision conditions (bottom). Both spectra were acquired at the calibration point for 2-BTPP. Deviation from Table 1 - 55%.

the effect of ion abundance variations in LSIMS has been discussed.' A rigorous mathematical treatment of the parent-to-daughter abundance variation has not yet been worked out, however. In practice, a similarity index (as described here) of 50 or below is sufficient for a match between an unknown and a standard compound. Using the standardization protocol described here, we can achieve that degree of similarity in most situations.

CONCLUSIONS

A calibration procedure for acquiring reproducible product ion taqdem mass spectra from parent ions generated by FAB/LSIMS on an EBqQ hybrid mass

spectrometer has been described. The protocol is simple to implement and is applicable to a large variety of instrument designs and ionization strategies. Run-to- run spectral reproducibility, based on the average difference percentage, is similar for both multiple and single collision conditions. Under both conditions, the day-to- day spectral reproducibility of the product ion spectra decreases as the masses of the model compounds deviate in either direction from the mass of the calibration compound. Additionally, the similarity between spectra obtained under the different pressure regimes increases as the masses of the model compounds become increasingly greater than the mass of the calibration compound.

Over the mass range examined here, the calibration compound, 2-BTPP, resulted in the best run-to-run reproducibility for the isomer, n-BTPP, and for the

342 3,3'-Dlhexadecyloxacarkyanine Cation

217 '"7 I

m/z

Figure 0. Comparison of the tandem mass spectrum of D-1125 acquired under multiple collision conditions (top) with the tandem mass spectrum acquired under single collision conditions (bottom). Both spectra were acquired at the calibration point for 2-BTPP. Deviation from Table 1 = 33%.


lowest mass compoupd, m-NBA. To obtain better spectral reproducibility for the other model compounds, other calibration compounds must be used, losing the advantage of a ‘universal’ calibration compound. For comparison of product ion tandem mass spectra obtained under different collision conditions, a calibration compound of mass lower than the mass of the analyte provides better spectral similarity. It should be possible to obtain both run-to-run reproducibility and better spectral similarity between spectra obtained under different collision conditions by careful selection of the calibration compound. To obtain run-to-run reproducibility, a calibration compound should be selected that has a mass (ml) that will satisfy the equation (ml/mz)”z = 0 3 , where i is an integer greater than zero and mz is the mass of the analyte. For i = 1 and 2, the mass of the calibration compound will be less than the mass of the analyte and equal to the mass of the analyte, respectively, providing better spectral similarity between spectra obtained under different collision conditions as well as better run-to-run reproducibility. As long as the above mass-related criterion is met, it should be possible to use any compound that produces a strong, stable signal and two suitable product ions in FAB/LSIMS as the calibration compound.

The pronounced nodal properties of quadrupoles contribute to the lack of reproducibility, as evidenced by the strong correlation between the observed pattern of reproducibility and the absolute deviation of the ratio (m,/m,)”z from the calibration maximum. Since nodes are significantly reduced in hexapole and octapole collision cells, the use of these configurations would be expected to improve greatly the spectral reproducibility. To confirm this presumed improvement in reproducibility we need to extend this work to instruments with an hexapole or an octapole collision cell. This work also needs to be extended to instruments with a quadrupole as the first mass analyzer. To these

ends, collaborators with such instruments would be wel- comed.

The current results were obtained with only one matrix, m-NBA, but the calibration compound, 2-BTPP, may be used to reach a calibration point in several other matrices, albeit at different collision energies. Thus far, no extensive comparisons of spectra obtained in a matrix different from that of the calibration compound have been made, but such comparisons would be a logical next step, and may reflect changes in the energetics of the ionization event medi- ated by different support matrices.

This method was adapted from a similar procedure for obtaining reproducible product ion EI tandem mass spectra described in a previous paper.4 The current work extends the application of the calibration point protocol to LSIMS MS/MS. In general, spectral reproducibility for LSIMS using this method was 15% worse than for EI-MS/MS based on the average difference percentage. In LSIMS, the measured abundances of individual ions can vary by as much as 10-15% from scan-to-scan compared with the relatively stable signals measured in EI.* Because the abundance of the parent ion selected for CID in LSIMS is more variable than in EI, it is not unreasonable to expect greater abundance variations for the product ions. Although the reproducibility is not as good as that obtained with more rigorous calibration procedures, it should be sufficient for the establishment and day-to-day use of a tandem mass spectral library for identification of unknown compounds.

Ackno wledgernent

We thank K. R. Mohan who performed the initial EI-MS/MS work and M. G. Bartlett for his invaluable assistance and guidance during the preliminary stages of this work.

REFERENCES

1 . P. H. Dawson and W. F. Sun, Int. J . Mass Spectrom. ton Phys.

2. S. Naylor and J. H. Lamb,Ana/. Chim. Acfa 241, 201 (1990). 3. R. I. Martinez, Rapid Commun. Mass Spectrom. 5, 245 (1 991 ). 4. K. R. Mohan, M . G. Bartlett, K. L. Busch, A. E. Schoen and N.

Gore, J. Am. SOC. Mass Spectrom. 5, 576 (1 994). 5. M. S. Kim, M . Rabrenovic and J. H. Beynon, Int. J. Mass

Spectrom. Ion Processes 56.7 1 ( 1 984).

6. W. DeLange, N. M. M. Nibbering, Int. J. Mass Spectrom. Ion

7. A. J. Alexander, E. W. Dyer and R. K. Boyd, Rapid Commun.

8. S. A. Testino, Jr. and K. L. Busch, Rapid Common. Mass

55,155 (1 983/84). Processes 86,287 (1 988).

Mass Spectrom. 3,364 (1 989).

Specrrom. 7, 251 (1993).

calibration point for liquid secondary ion massspectrometry tandem mass spectra measuredwith an ebqq...

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