Anal. Chem. 1995, 67,1696-1704
Improved Collisionally Activated Dissociation Efficiency and Mass Resolution on a Triple Quadrupole Mass Spectrometer System Bruce A. Thomson,* D. J. Douglas, Jay J. Cow, James W. Hager, and Charles L. Jolliffe SCIEX, 55 Glen Cameron Road, Thomhill, Ontario, Canada L3T lP2
CollsionaUy activated dissociationei€tciencyand fragment ion mass resolution have been characterized on a triple quadrupole mass spectrometer equipped with an enclosed collision cell which is operated at relatively high pressure. The higher pressure results in improved efficiency (and therefore improved ion transmission) and better mass resolution than has been obtained previously with collision cells operated at lower pressure. Efficiencies of 30-50%, measured as the fractions of original precursor ion in Q l which are detected as resolved fragment ions, are typical for singly charged ions, compared with 5- 10%achieved previously. Mass resolution of fragment ions is improved to the point that the isotopes from quadruply charged fragment ions can be resolved. A typical triple quadrupole mass spectrometer (MS/MS) system' consists of two resolving quadrupole mass filters (Q1 and Q3) separated by another quadrupole assembly (Q2) into which a collision gas can be introduced to allow collisionally activated dissociation (CAD) in the radio frequency (rf) field. In an MS/ MS experiment, the first quadrupole is used in a mass resolving mode to select the precursor ion, and the thiid quadrupole is used in a mass resolving mode to provide mass analysis of the resulting fragment ions. In some variations, octapole or hexapole assemblies are used in place of a quadrupole for the central collision region. In a single MS mode of operation, the collision gas is turned off, ions are resolved with either Q1 or Q3 (with the other quadrupole operated in an rf-only or total ion mode), and ions are detected at the exit of Q3. For the purpose of this discussion, we will assume that single MS operation is always performed with Q1, although the results will apply approximately to the case where Q3 is used for single MS. Comparing operation in an MS/ MS mode to operation in a single MS mode, there are two significant factors which may lead to the loss of some of the ions: (1) ions are scattered from the ion path by the collision gas in Q2 and (2) ions which reach the entrance to Q3 are transmitted through Q3 with less than 100%efficiency because Q3 is operated in a mass resolving mode. Although operation in the MS/MS mode always results in less sensitivity, in many cases the gain in signal-to-noiseachieved due to the reduction in background noise, and the additional specificity offered by the double stage of mass analysis, result in far better detection limits and better quantitation than operation in a single MS mode. (1)Yost, R A;Enke, C. G. Anal. Chem. 1979, 51,125lA-12644,
1696 Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
Very few quantitative analyses of the transmission characteristics of triple quadrupole mass spectrometers have appeared in the literature. Dawson et al.2 showed efficiency measurements of one version of a collision cell in which the gas is introduced in a free jet across the axis of the quadrupole. These investigations suggested that scattering losses were minimal in the collision cell; however, the experiments were limited to low-mass (200%and EM=50% for reserpine, for example) are closer to those reported for the ion trap in ref 4, suggesting that at the higher collision cell pressure which we employ, the ion confinement and transmission efficiency of the triple quadrupole begin to approach that of the ion trap. This does not obviate the advantage of the “tandem-in-time”aspect of the ion trap over the “tandem-in-space” aspect of the triple 1700 Analytical Chemisty, Vol. 67, No. 10, May 15, 1995
a d
c
.0001 0 -20
-10 0 10 20 Q3 Rod Offset (V)
10 1
1
b,
-20
30
-10
0 10 20 0 3 Rod Offset ( V )
I
30
Figure 5. Stopping curves for the precursor ion m/z 609 (0)and fragments m/z448 (M)and 195 (0) at (a) CGT = 1.5 x 10i5/cm2 and (b) CGT = 4 x loi5/ cm2.Q2 rod offset voltage, 0 V; collision energy, 30 eV.
quadrupole4in a scanning mode of operation, which leads to a potentially much higher efficiency of ion detection. However, for targeted compound analysis, where only a few ions are monitored, the efficiencies which we observe suggest that the advantage in sensitivity of the ion trap over the triple quadrupole is less clear. The second significant advantage of operating at a high collision cell pressure is that the ion kinetic energy distribution of ions exiting from Q2 is considerably smaller, which contributes both to the enhanced transmission through Q3 and to the ability to obtain improved mass resolution on the fragment ions. This is illustrated in Figure 5, which shows stopping curves for two of the major fragment ions from reserpine (mlz 195 and 448) and for the survivor precursor, obtained by varying the Q3 rod offset voltage? In the example shown, ions enter Q2 with a kinetic energy of -30 eV. At a CGT of 1.5 x 1015/cm2,the stopping curves for the three ions show different average ion energies and considerable energy spread. These mass-dependent energies are well known in triple quadrupole mass spectromet~y.~~ When CGT is increased to 4 x 1015/cm2,however, the stopping curves become almost identical and the energy spreads are significantly reduced. This assists in achieving both high transmission and good mass resolution (as discussed later) through Q3 without the necessity of scanning the Q3 rod offset with mass (see ref 14 for a discussion of the merits of scanning the Q3 rod offset). Although the use of Q3 in a resolving mode as an energy stop does not provide sufficient field uniformity to measure with great accuracy the kinetic energy of the ions which exit from Q2, it is clear that ions must emerge with very little kinetic energy, typically retaining little or no residual component of the injection energy into Q2. This observation follows from the fact that all ions are effectively stopped by a Q3 rod offset potential equal to that of Q2, independent of the collision energy. It is less clear what factors ensure that ions actually emerge from Q2 rather than simply coming to rest in the middle. (14)Shushan, B.; Douglas, D. J.; Davidson, W. R; Nacson, S. Int. J. Mass Spectrom. Ion Phys. 1983,46, 71-74.
Table 1. Maximum Values of Collection Efficiency (&) and MSMS Efficiency (EM)for Various Singly Charged Parent Ions of Ammonium Adducts of Poly(propy1ene glycol)'
m/z
Ec (%I
EM(%)
384 500 907 1545 2010
61 61 210 410 910
32 34 28 43 a7
Values of collision energy and CGT at the maximum are as follows: m/z 384, 20 eV, CGT = 4.3 x 1015/cm2; m/z 500, 20 eV, CGT = 4.3 x 1015/cm2; m/z 907, 25 eV, CGT = 4.3 x 1015/cm2; m / z 1545, 37 eV, CGT = 5.6 x 1015/cm2;and m/z 2010, 55 eV, CGT = 5.6 x 10vcm2. (I
A phenomenological consideration of the parameters which affect Ec and EMmight be expected to reveal trends with precursor ion mass (or m / z ) . As Douglas and French demonstrated, the effects of collisional focusing are greater for heavier (i.e., higher mass) ions. This should lead to an increase in both EC and EM with precursor ion mass, particularly in cases where the highmass precursor forms relatively high mass fragments. However, with Q3 operated at unit mass resolution, the transmission of Q3 decreases with increasing mlz. This factor should result in EC increasing with mass (due to POdecreasing), while EMdecreases (Q3 transmission affecting the numerator but not the denominator in the expression for E d . In order to elucidate the trend, a series of ions of various molecular weights from poly(propy1ene glycol) (PPG) was studied. The precursor ions consist of ammonium ion adducts of the form OH(C3H7O)"NH4+, all of which fragment to a series of low-mass ions of m / z 59, 117, 175, 233, 291, etc. Precursor ions of m / z 326, 500, 907, 1545, and 2010 were fragmented over a range of collision energies and CGT values sufficient to determine the maximum values of Ec and EM. Since the major fragment ion masses are the same for all precursor ions ranging from m/z 326 to 2010, the dominant variable should be the precursor ion mass. Note that these loosely bound adduct ions are not necessarily good models of the behavior of other high-mass ions (peptides, for example), which may require higher collision energies for fragmentation; however, the homologous series provides a convenient ladder of similar species in which the major variable is precursor ion mass. The results, shown in Table 1, reveal a nearly monotonic trend of increased efficiency at higher mlz for both EC and EM. This may be at least partly due to the increase in the mass ratio between precursors and fragments as the precursor mass increases, reflected as the increased transmission of the lower mass (hence lower resolution) fragments compared to the higher mass (hence higher resolution) precursor. An additional factor is the widely different values of the Mathieu q parameter2,6in Q2 for a highmass precursor and a low-mass fragment, which must simultaneously be confined. Q2 is coupled to Q3 at approximately 33% of the Q3 rf level, so that when Q3 is set to transmit m / z 59, the q of m/z 2010 will be -0.008. The low q value for the precursor might be expected to result in poor confinement in Q2, and hence poor sensitivity toward low-mass fragments formed from highmass precursors. This effect cannot be independently evaluated for the PPG ions, since the distribution of fragments among the values listed above may be somewhat different for different
precursors. However, the relatively high efficiency observed for m / z 2010 suggests that fragmentation may occur close to the entrance of Q2, before destabilization of the precursor in 8 2 becomes significant. It is also revealing to consider the effect of charge state on the apparent CAD efficiencies. In the most common instances of multiply charged (positive) electrospray ions, the multiple charges consist of protons attached to various basic sites of the molecule. Collisional activation of a multiply charged ion, followed by unimolecular decomposition, can readily lead to the formation of two ions from one (for example, a singly and a doubly charged ion from a triply charged precursor). If one or both of the products are multiply charged, further collisions can lead to more ion multiplication. In the extreme case, a single precursor ion with a charge state n could be fragmented to n singly charged ions, which would result in an increase in the total number of ions from each single precursor by up to a factor of n. More typically, one might expect some (but not complete) conversion of multiply charged precursor ions to fragments with lower charge states and therefore expect to see a net increase in the number of ions leaving the cell compared to ions entering the cell. The degree of conversion to lower charge states will, however, be dependent upon the details of the fragmentation process. In some cases, loss of a neutral from a multiply charged precursor will still be a preferred route. The pulse counting detection system employed here, which detects and counts pulses from individual ions, allows indirect observation of any multiplication effect from multiply charged parents. Ions are detected independent of charge state, in contrast to a Faraday cup measurement, which would record total charge rather than number of ions. An analogue detector, in which the detected signal is proportional to the number of primary electrons formed on the first conversion dynode, might be expected to show a response somewhere in between total ion rate and total charge rate. The ion multiplication effect has been explored by fragmenting the precursor ions of four d ~ e r e ncharge t states of the tetrade capeptide renin substrate (DRVYIHPFHLLVYS), from (M J3) to @ 4H)+4 l (corresponding to precursor ion m / z values of 1759,880,587,and 440, respectively). RepresentativeCAD spectra of the four are shown in Figure 6, where collision energies and CGT values have been adjusted to provide spectra with fragments throughout the mass range. Close examination of the spectra shows that in each case (except for the singly charged), there is a signifcant amount of charge state reduction in the fragments compared to the precursor, and thus one might expect to see a greater apparent efficiency for the higher charge state parents than for the lower charge state parents. Table 2 shows the values of EC and EMmeasured for each of the four precursor ions under these conditions. The unexpected observation that EC appears to decrease with increasing charge state is probably due more to the fact that Po decreases with increasing mlz (as discussed above) than to any effects related to charge state. [Note here that while m / z decreases with increasing charge state, the mass m remains constant; the only effect on collisional focusing should be due to the increased charge, which appears to somewhat increase the collision cross section.15] EM,on the other hand, appears relatively constant with charge state, although higher than is the case with reserpine and the PPG precursor ions (compare with Table 1).
+
+
+
(15) Covey, T. R;Douglas, D. J.J. Am. SOC.Mass Spectrom. 1993,4,616-623.
Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
1701
a)
Q
=-5
l,o
100 50 0
al
'
...A
583 640 746784
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I
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,
,
.
136
loo 86 50 0
d)
1029
-1 200
400
600
+
+
800
1000
mlz
1200
1400
1600
+
Figure 6. CAD spectra of (a) (M H)+ ( d z1760), (b) (M 2H)*+ (dz880), (c) (M + 3H)3+( d z 5 8 7 ) , and (d) (M 4H)4+( d z 4 4 0 ) for renin substrate. CAD conditions: mlz 1760, 55 eV; d z 880, 60 eV; mlz 587, 45 eV; mlz 440, 40 eV. CGT = 4 x 10i5/cm2 in all cases. Base peak intensities are (a) 8.7 x lo4, (b) 8.8 x lo4 (c) 1.2 x lo5, and (d) 5.8 x lo4 counts/s. Table 2. Collection Efficiency ( 4 ) and MSMS Efficiency (€4for (M H)+ (nJz1760), (M 2H)*+ (nJz 8801, (M 3H)3+(nJz u)7), and (M 4H)'+ (nJz 440) for Renin Substrate.
+
+
a
+
+
m/z
charge state
Ec (%I
EM (%)
1760 880 587 440
1 2 3 4
830 890 430 220
89 70 99 97
MS/MS conditions: m/z 1760,55 eV m/z 880,60 eV; m/z 587,45 x 10i5/cm2in all cases.
eV and m / z 440, 40 eV. CGT = 4
The apparent lack of correlation between charge state and efficiency under nominal CAD conditions shows that the situation is more complicated; the effects of mass resolution and energy may mask any trends associated purely with ion multiplication. More detailed studies of the complementaryfragments in the CAD of multiply charged ions are required. Tang and Boyd16 have commented on the relative d ~ e r e n c e sin intensities between y' and b series of complementary singly charged ions in the fragment spectra of doubly charged tryptic peptides. They attributed the difference to the stability of the y' ions to further dissociation relative to the b ions and showed in the case of a small peptide that the latter more readily dissociated to smaller fragments. This suggestion, if also true for the example explored here, would only account for the present results if such lower mass fragments were themselves discriminated against in the collision cell. A better understanding of the sequential fragmentationprocesses, and the transmission of very low mass ions through the collision cell, is required in order to more clearly elucidate the overall charge and mass budget of the CAD process in the triple quadrupole collision cell. Resolution. Very little attention has been paid in the literature to the mass resolution of fragment ions in triple quadrupole systems. In many literature publications of triple quadrupole applications, the mass resolution of fragment ions is not stated, nor is it obvious from the data, which have often been centroided. In other cases, it is stated that Q3 is operated under less than (16) Tang, X.-J.; Boyd, 657.
R IC Rapid Commun. Mass Spectrom. 1992,6, 651-
1702 Analytical Chemisfry, Vol. 67, No. 10, May 15, 1995
unit mass resolution.6 It has been our experience that unit mass resolution on fragment ions greater than a few hundred amu in mass is dficult to achieve at the relatively low collision gas pressures typically used in the past, due to the fact that the CAD process leads to fragment ions which have mass-dependent kinetic energies and significant energy spreads.14 One solution to this problem has been to scan the Q3 rod offset as a function of fragment ion mass, in an effort to slow the ions to the same energy. Shushan et al.14 empirically found that a parabolic ramp with mass was most effective; however, the effectiveness of this approach was demonstrated only for a relatively low mass precursor ion of m / z 223. In general, fragment ion energies are only approximately known, so it is not possible to define a scan function for the Q3 rod offset which is optimum for all situations. In any event, a relatively wide energy spread associated with any particular fragment ion does not allow full control over the kinetic energy of the ions entering Q3. As discussed earlier and shown in Figure 5, when Q2 is operated at a target gas thickness of greater than about 3 x 1015/ cm2, all fragment ions exit Q2 at the same kinetic energy, independent of mass. A constant potential difference between Q2 and Q3 (typically 5 V) results in a constant energy of 5 eV in Q3 (for singly charged ions). In addition, the sharp stopping curves shown in Figure 5 suggest that the ions have relatively little kinetic energy spread. Defining the width of the energy distribution as twice the difference between the average value (midpoint on the stopping curve) and the value where the intensity has decreased to lo%,the absolute spread in energies shown in Figure 5 at the higher CGT is -1 eV for all three ions. Since the resolution of a quadrupole operated in a constant peak width mode is proportional to n2 (cf. ref 13), where n is the number of cycles in the rf field, it is simple to show that in the case where all ions have the same kinetic energy independent of mass (as shown in Figure 5 , for example), the resolution will increase proportional to mass, and therefore the peak width will be constant, independent of mass and charge state. Constant peak width across the mass range can therefore be achieved without scanning the Q3 rod offset as a function of fragment ion mass. The narrow energy distribution means that there are few high-
1001
/I
n
mh
100
200
300
400
500
600
700
Figure 9, Fragment ion mass of d z 746 doubly charged ion from d z 4 4 0 quadruply charged precursor ion of renin substrate, showing relative intensities at four different resolutions. Collision energy, 28 eV; CGT, 4 x 1015/cm2.
mlz
Figure 7. CAD spectrum of m/z 609 precursor ion from reserpine, showing resolution of fragment ions across the mass range. Collision energy, 35 eV; CGT, 4 x 1015/cm2.
n
"
200
+3
400
600 mh
600
An
+a
1000
1200
+
Figure 8. CAD spectrum of (M 4H)4+ (mlz440) from renin substrate at high fragment ion resolution, showing separation of the isotopes for fragment ions of four different charge states. Collision energy, 28 eV; CGT, 4 x lOI5/cm2.
energy ions (which contribute to peak tailing on the low-mass side) in Q3. All spectra shown previously were obtained with a constant 5-V difference between the Q2 and Q3 rod offsets. Typical operating resolution in the MS/MS mode under this condition is shown in Figure 7 for reserpine, where the peak shape of the fragment ions is shown in segments of the spectrum. This peak width of about 0.7 amu independent of mass is typical of unit resolution conditions for fragment ions and is representative of the resolution under which all of the CAD efficiency data described in the previous section were acquired. Significantly higher resolution can be achieved by decreasing the potential difference between Q2 and Q3 to 3 V in order to further slow the ions down, and increasing the dc/rf ratio of Q3 (which increases the quadrupole resolution). An example of the resolution which can be achieved on fragment ions is shown in Figure 8. The CAD spectrum of the quadruply charged precursor ion from renin substrate, at m / z 440, reveals fragment ions with charge states ranging from 1 to 4, and operation under conditions of high resolution in Q3 allows the isotopic peaks of the fragment ions to be resolved. For the purpose of demonstrating the
resolution by showing separation between the isotopes, the Q1 resolution has been decreased in order to pass the entire isotopic cluster of the precursor through Q1. As the insets in Figure 8 clearly show, charge states of up to at least 4+ can be easily identified by the spacing of the isotopes. The width of the peak shown at m / z 745, a doubly charged fragment, is 0.135 amu at half-height, which gives a resolution M/AM on this fragment of just over 5500. Resolution this high has not been previously reported for fragment ions in a triple quadrupole system. The higher resolution is achieved at the expense of sensitivity; however, the penalty is not severe. For example, Figure 9 shows how the peak height varies with resolution for the doubly charged fragment at m l z 745. There is less than a factor of 3 difference in peak height between a resolution where the isotopes are just barely separated at the top and a resolution where the isotopes are well separated with better than a 50%valley. In a more extreme example, a comparison of the peak height at unit resolution for singly charged ions, where the peak width at halfheight is -0.7 amu and the isotopes of a 4+ fragment are not separated,to the peak height at a resolution where the 43-charge state fragment ion can be identified by its isotopes, shows approximately a factor of 6 difference for the m / z 436 fragment ion from renin substrate. The ability to operate under the high-resolution conditions demonstrated above can be very useful for identifying the charge state of the fragment ions and thus the true masses of the fragments. Charge state identification by this means is facilitated by reducing the Q1 resolution in order to pass the full isotope cluster of the precursor, as shown in Figure 9 for the quadruply charged precursor of renin substrate. CONCLUSION
Characterization of the transmission efficiency of a triple quadrupole mass spectrometer system for precursor ion masses up to 2000 amu has shown that high efficiency can be achieved under multiple collision conditions. Performance in terms of efficiency relative to the Q1 single MS intensity appears if anything to increase rather than decrease as the precursor ion mass increases. In addition, the multiple collisions assist in slowing the ions so that a relatively monoenergeticbeam enters Q3. This allows the fragment ions to be well resolved in Q3, to the extent that isotopic peaks from quadruply charged ions can be separated. Analytical Chemistry, Vol. 67, No. 10, May 15, 1995
1703
The overall result is that operation in the high-pressure regime explored in this paper provides signilicantly higher mass resolution along with significantly higher sensitivity than does operation at lower pressures. While all of the factors which lead to the improved performance are not understood, the data are generally consistent with the concept of collisional focusing under multiple collision conditions in an rf field. The implication from this work is that operation at high target gas thickness is advantageous from the point of view of both sensitivity and mass resolution, factors which are of prime importance to analytical applications of triple quadrupole mass spectrometry. The implications for maximizing “chemical information” in the CAD spectra have not been systematically explored. Our experience to date suggests that a similar degree of fragmentation is achieved with a lower collision energy at high target gas thickness than is required at the lower target gas thickness. Instrumental factors in the triple quadrupole which result in high collection efficiency under multiple collision conditions thus appear to move us partly toward operation in the very low energy/very high collision number regime, which is currently accessible only in the ion trap. The limitation may be that in the
1704 Analytical Chemistry, Vol. 67,No. 10,May 15, 1995
triple quadrupole, energy can be controlled only by accelerating the ions into the entrance of the collision cell (ions thereafter slowing down through collisions in the field-free region), whereas in the ion trap, energy can be continuously pumped into the precursor ions by resonant excitation. The attempt to observe a very signi6cant increase in efficiency due to ion multiplication when multiply charged parents are fragmented was unsuccessful. Examination of the spectra for the example studied suggested that complementary mass pairs were not present in equal abundance, which could explain the observed efficiencies. Resolution of this anomaly will require a more careful exploration of the formation of complementary mass fragments, their continement and transmission efficiency into Q3, and the effect of further fragmentation to very low mass fragments. Received for review September 30, 1994. February 17, 1995.@
Accepted
AC9409694 @
Abstract published in Advance ACS Abstracts, March 15,1995.
