Electrospray Ion Trap Mass Spectrometry

Transferability of the Electrospray Ionization Efficiency Scale between Different Instruments. Jaanus Liigand , Anneli Kruve , Piia Liigand , Asko Laa...
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Anal. Chem. 1999, 71, 1441-1447

Improvements in LC/Electrospray Ion Trap Mass Spectrometry Performance Using an Off-Axis Nebulizer Robert D. Voyksner* and Heewon Lee†

Analytical and Chemical Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709

Charged residues from the electrospray process have been hypothesized to limit the sensitivity and dynamic range of an ion trap mass spectrometry operation. Incorporation of an off-axis nebulizer (positioned 90-95° from the sampling orifice) was found to drastically reduce the detrimental effects caused by the charged particles or droplets compared to typical on-axis nebulization configurations (spraying 10-20° from sampling orifices). The off-axis nebulizer reduced total ion currents that enter the ion trap (through the reduction of charged residues) by a factor of 5-7 while resulting in an increase of analyte [M + H]+ signal by a factor of 6 compared to an on-axis sprayer at flow rates of 20 µL/min. At higher flow rates (e.g., 800 µL/min) these enhancements are more evident. At flows greater than 200 µL/min, off-axis nebulization reduced total ion current that enters the ion trap by a factor of 30 and resulted in a factor of more than 20 increase in [M + H]+ signal relative to on-axis nebulization. Incorporation of the off-axis nebulizer improved the detection limit and precision for determination of dihydroxyvitamin D3 in plasma compared to on-axis nebulization. The LC/MS/MS detection limits obtained for the offaxis nebulizer on the ion trap was within a factor of 2 from the detection limit determined by the triple quadrupole. The relative standard deviation of the dihydroxyvitamin D3 determination was less than 8% for both off-axis ion trap and triple-quadrupole determinations.

Two mechanisms have been proposed for the formation of ions by electrospray. The first, is ion evaporation, which results in analyte ion desorption from a droplet due to high-field strength generated by charged droplets.1-5 The other relies on Rayleigh fissions to form very small charged droplets followed by solvent evaporation to result in a gas-phase analyte ion.3,5-7 Improvements to the nebulization and desolvation processes brought by pneu* Corresponding author: (Tel) (919) 541-6697; (fax) (919) 541-7208; (e-mail) [email protected]. † Current address: Ar Qule, 200 Boston Ave., Medford, MA 02155. (1) Iribarne, J. V.; Thomson, B. A. J. Chem. Phys. 1976, 64, 2287-2294. (2) Thomson, B. A.; Iribarne, J. V. J. Chem. Phys. 1979, 71, 4451-4463. (3) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (4) Fenn J. B. J. Am. Soc. Mass. Spectrom. 1993, 4, 524-535. (5) Kebarle, P.; Yeunghaw H. In Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Application; Cole, R. B., Ed.; John Wiley & Sons: New York, 1997; pp 1-63. 10.1021/ac980995s CCC: $18.00 Published on Web 03/04/1999

© 1999 American Chemical Society

matic nebulization, additional drying gas, and heat have increased the range of solvents and solvent flow rates that can be handled by the electrospray technique.8 Still, variations in the droplet size and charging can result in charged species that do not yield analyte ions due to insufficient field strengths to result in ion evaporation. Also droplets that have desorbed analyte ions remain in the API chamber as charged droplets or charged residues.9 These charged droplets that are void of analyte can interfere with the ion trap mass spectrometry analysis, as is evident in spectra that exhibit high background noise and spike noise as well as contaminate the optics in the electrospray transport region. The charged residues, propelled by their initial momentum from the pneumatic nebulization and brought into the API interface by viscous gas drag through the sampling orifice, are not easily filtered by quadrupole mass analyzers. Most manufactured electrospray interfaces attempt to negate the effects of the charged residues by using off-axis nebulizers, have an arrangement of offaxis skimmers or skimmer capillary sampling orifices, or use a shield in front of the sampling orifice.10,11 Off-axis detection and pulse counting detection also aid in the reduction of noise created by charged residues. The rapid emergence of atmospheric pressure ionization (API) techniques with ion trap mass spectrometers (ITMS)12 warrants investigations into the effect charged residues have on the performance of this mass analyzer. It has been demonstrated that charged species play a significant role in reducing sensitivity and dynamic range of the trap, through space charging,13-15 and it is theorized that charged residues may degrade trap performance in a similar manner. There are reports in the literature where long (6) Dole, M.; Mack, L. L.; Hines, R. L.; Mobley R. C.; Ferguson L. D. Alice, M. B. J. Chem. Phys. 1968, 49, 2240-2249. (7) Schmelzeiser-Redeker, G.; Buttering L.; Rollgen, F. W. Int. J. Mass. Spectrom Ion Processes 1989, 90, 139-150. (8) Huang E.; Wache, T.; Conboy, J. J.; Henlon, J. D. Anal. Chem. 1990, 62, 713-725. (9) Kostiainen, R.; Bruins A. P. Rapid Commun. Mass Spectrom. 1996, 10, 1393-1399. (10) Goodley, P. C.; Fischer, S. M.; Gourley, D. L. Self-Generating Ion Device for Mass Spectrometry of Liquids. U.S. Patent, 5,559,326, 1996. (11) Hopfgartner, G.; Wachs, T.; Bean, K.; Henion, J. Anal. Chem. 1993, 65, 439-446. (12) McLuckey, S. A.; Van Berkel, G. J.; Goeringer, D. E.; Gish, G. L. Anal. Chem. 1993, 66, 737-743. (13) March, R. E. J. Mass Spectrom. 1997, 32, 351-369. (14) Mathurin, J.-C.; Gregoire, S.; Brunot, A.; Tabet, J.-C.; March, R. E.; Catinella, S.; Traldi, P. J. Mass Spectrom. 1997, 32, 829-837. (15) Doroshenko, V. M.; Cotter, R. J. J. Mass Spectrom. 1997, 32, 602-615.

Analytical Chemistry, Vol. 71, No. 7, April 1, 1999 1441

Figure 1. Schematic of the LC/electrospray ion trap MS system showing the configuration for the off-axis and on-axis nebulizers evaluated in this paper.

desolvations are implemented after ion introduction in the trap,16 and the use of off-axis capillary-skimmer ion transport geometry appears to reduce the charged residues and improve an ion trap signal-to-noise (S/N) level. Detection of lipophilic vitamins such as vitamin D3 and their analogues proves to be a challenge due to the low levels that result in physiological effects and the need to have accurate and specific analytical methods for their detection. Due to the absence of acidic or basic sites on vitamin D3, derivatization followed by LC/ electrospray MS,17-19 LC/electrospray MS/MS,20 and LC/negative chemical ionization MS on a particle beam interface21 has been reported to achieve nanogram per milliliter sensitivity levels. Since derivatization requires an additional step in the method and does not provide direct evidence for the species of interest, nonderivatization methods that meet the required nanogram per milliliter sensitivity are required. LC/atmospheric pressure chemical ionization (APCI)-MS and MS/MS techniques have been reported for the determination of underivative vitamin D3.22 Typically APCI is the ionization technique of choice since vitamin D3 has no acidic or basic sites to form precharged ions which would enhance electrospray sensitivity. The APCI process only generates an [M + H - H2O]+ ion for vitamin D3; no [M + H]+ ion is detected. However, the detection of various analogues of vitamin D3 can be hindered by APCI due to additional thermal fragmentation by APCI such as sequential losses of water.22 While electrospray does (16) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1295. (17) Ishigai, M.; Ishitani, Y.; Kumaki, K. J. Chromatogr., B 1997, 704, 11-17. (18) Wilson, S. R.; Lu, Q.; Tulchinsky, M. L.; Wu, Y. J. Chem. Soc. Commun. 1993, 664-665. (19) Wilson, S. R.; Wu, Y. J. Am. Soc. Mass Spectrom. 1993, 4, 596-603. (20) Yeung, B.; Vouros, P.; Siu-Caldera, M.; Reddy, G. Biochem. Pharmacol. 1995, 49, 1099-1110. (21) Wang, K.; Davis, P. P.; Crews, T.; Gabriel, L.; Edom, R. W. Anal. Biochem. 1996, 243, 28-40. (22) Seipelt, C. T.; Caldwell, B. E.; Gilliland, D. L. Presented at the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palms Springs, CA, June 1-5, 1997; p 170.

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not yield a molecular species for an analogue such as dihydroxyvitamin D3, no additional fragmentation is observed beyond the initial loss of water. This paper demonstrates the advantages offered by an off-axis pneumatic nebulizer in an ITMS system. The performance as determined by sensitivity, signal-to-noise ratio, precision, and ruggedness of the off-axis nebulizer will be compared to conventional on-axis nebulizers for the determination of dihydroxyvitamin D3 in plasma. EXPERIMENTAL SECTION API-Ion Trap MS. A Finnigan MAT ion trap mass spectrometer (San Jose, CA) housed in a differentially pumped singlequadrupole vacuums manifold was interfaced with a modified Analytica of Branford API interface (Branford, CT), as shown in Figure 1. The main modification of interest to this paper is the use of an off-axis nebulizer to allow the introduction of the sample 90-95° from the sampling orifice (capillary entrance). The system was designed to retain the ability to place the nebulizer on-axis (10-20° from the sampling orifice) for direct comparison of performance using the same API interface and needle assembly. The system used four stages of pumping to maximize the ion flux into the ion trap. Stage 1 was pumped with two Edwards E2M18 pumps, stage 2 with an Edwards E2M30 pump, Stage 3 with a 520 L/S turbo pump, and stage 4 with a 170 L/S turbo pump. The resulting pressures for stages 1-4 under API operation without helium going to the ion trap are about 2, 0.1, 10-4, and 10-6 Torr (uncorrected gauge readings), respectively. Under normal API-ion trap MS operation, helium was added directly into the trap via a Teflon tube to achieve a stage 4 ion gauge pressure of 1.5 × 10-4 Torr (uncorrected ion gauge reading). Taking into account that the trap is relatively tight (Teflon spacers seal the openings between the ring electrode and end caps) and the gauge response of helium vs nitrogen, it is estimated that the pressure inside the trap is in the 2-5-mTorr range.

The ITMS was operated using the Teledyne Apogee (Mountain View, CA) system to perform full-scan MS and tandem MS (MS/ MS) experiments. A full-scan MS scan function would have a period for gating ions into the ion trap (0.2-20 ms) followed by a mass scan. The ions were gated into the trap at ∼800 Vpp (1.1 MHz) on the ring electrode. The scan from m/z 80 to 450 was accomplished using the mass-selected instability mode of operation where the rf amplitude was ramping at 5000 Da/s on the ring electrode ejecting each ion from the trap at a qz of 0.908. A linear voltage ramp on the end caps (1 Vpp per 100 Da with a 0.1V offset) at 0.65 MHz was applied during the voltage scan on the ring electrode to improve the resolution of the acquired mass spectra (pump scan). Ions were detected using an electron multiplier positioned on-axis after the exit end cap of the ion trap. The MS/MS scan function has a period for gating ions into the trap (1-200 ms) together with mass isolation, collision-induced decomposition (CID), and product ion mass scan. The gating of ions and the mass scan are similar to the previously discussed full-scan function. Simultaneously to gating, isolation of the desired target ion at m/z 399 ([M + H - H2O]+ ion from dihydroxyvitamin D) was accomplished by filter noise fields (FNF)23-25 to eject all but the desired ions from the trap. After isolation, a low voltage (0.75-0.85 Vpp) (at a qz value of 0.14) is applied to the end cap for 15 ms at the resonance frequency for the mass-isolated ion in order to generate the CID product ion spectrum. The product ion spectrum was accomplished by a mass-selective instability scan (such as described above), by scanning the voltage on the ring electrode at 5000 Da/s over the mass range of m/z 200-450. The API nebulizer (both the on-axis and off-axis nebulizers) used a 33-gauge stainless steel tube to transfer liquid from the LC or a syringe pump to the API chamber. Pneumatic nebulization with nitrogen (80 psi) and a counterflow of nitrogen (6-8 L/min) heated to 290 °C aided in the nebulization and desolvation of the introduced liquid from the off-axis or on-axis nebulizer. The API (electrospray) interface was typically operated using the following voltage settings: needle 0 V (grounded), Vcylinder at -5 kV, Vend plate at -4 kV, Vcapillary entrance at -4.5 kV, capillary exit at 100 V, skimmer 1 at 30 V, and skimmer 2 at 20 V. An octopole rf ion guide was used to transmit ions from skimmer 2, through a stage of pumping, and into the entrance end cap of the ion trap. The rf ion guide used cylindrical rods (97 mm × 1.25 mm) with an r0 of 2.17 mm. It was operated at 1.8 MHz at 600-900 Vpp. with a 4-V bias (HP 3110B function generator and ENI power amplifier model A2525PA-00051). The rf voltage was used to determine the low-m/z cutoff of the ion guide. The end of the rf ion guide was located 3 mm from the orifice of the entrance end cap of the ion trap. A cylindrical electrode (gate) of 12 mm in length mounted around the ion guide and extending ∼0.5 mm to the entrance orifice of the end cap was used to control the entry of ions into the ion trap and for the storage of ions in the rf ion guide. The gate, controlled through the Teledyne electronics, allowed ions to enter the trap for up to 200 ms. The gate was toggled to 0 V during ion (23) Kelley, P. E. Mass Spectrometry Method Using Filtered Noise Signal. U.S. Patent, 5,206,507, 1993. (24) Kelley, P. E. Mass Spectrometry Method Using Notch Filter. U.S. Patent 5,134,286, 1992. (25) Kenny, D. V.; Callahan, P. J.; Gordon, S. M. Rapid Commun. Mass Spectrom. 1993, 7, 1086-1089.

introduction. During the rest of the scan function, the gate was toggled to +60 V. This voltage was also optimal for the storage of ions in the rf ion guide. The end cap and nebulizer spray currents were measured with a high-resistance current meter (Hewlett-Packard model 4329A) on the 10-12 A setting. The end cap currents (an entrance hole plugged with metal foil) were measured under normal operating conditions with the gate disabled and set to a low voltage to transmit ions to the trap. The nebulizer spray currents were measured with a needle electrode held 2.5 cm from the nebulizer at angles from 0 to 150° from the axis of the nebulizer. Samples and Solvents. The initial comparison of capabilities between off-axis and on-axis nebulizer on the ion trap was performed using infusion of solvent standards. The standard compounds analyzed included lincomycin, adenosine, and betaine (Sigma, St. Louis, MO). These standards were dissolved in 50/ 50 acetonitrile (ACN)/water with 1% acetic acid, (lincomycin at a level of 2 ng/mL while betaine and adenosine were added from 2 to 100 ng/mL, and these test solutions were either infused into the API-ion trap MS at flow rates ranging from 20 to 800 µL/min. The solvent was delivered using a syringe pump (Orion Research Inc., Boston, MA). Extraction and Separation Conditions for Dihydroxyvitamin D3. A 1-mL volume of rat plasma spiked with dihydroxyvitamin D3 from 0.3 to 30 ng/mL was mixed and vortexed for 2 min with an equal volume of chlorobutane. The dihydroxyvitamin D3 was partitioned into the chlorobutane phase, and the chlorobutane was removed from the mix and evaporated to dryness under nitrogen at 20 °C. The extract was reconstituted with 50 µL of 70% acetonitrile in water, and 10-20 µL of the extract was injected for the LC/MS analysis. The LC separation was performed with a Hewlett-Packard model 1090 (Palo Alto, CA), on a Zorbax XDB C18 2.1 × 50 mm (Mac-Mod, Chadds Ford, PA) using isocratic conditions of 70% ACN with 1% acetic acid at a flow rate of 0.3 mL/min. These conditions resulted in a retention time for dihydroxyvitamin D3 of 1.9 min. Single-Quadrupole API-MS. The single-quadrupole electrospray determination of lincomycin for on-axis and off-axis nebulization was performed using the above conditions (described for samples and solvents) on a Hewlett-Packard 5989 with a HewlettPackard API interface fitted with a hexapole ion guide (Analytica of Branford). The instrument was tuned and optimized for the transmission of the [M + H]+ ion of lincomycin at m/z 407 by infusing a 2 ng/µL solution of the antibiotic into the system. The LC/MS analysis was performed using selected-ion monitoring, detecting m/z 407 for lincomycin with a dwell time of 300 ms. Identical conditions were used for both the off-axis and on-axis determinations on the LC/MS system. Triple-Quadrupole API-MS/MS. The triple-quadrupole work was preformed on a Micromass Quarto II (Manchester, UK) with the Hewlett-Packard 1100 LC system. The identical dihydroxyvitamin D3 plasma extracts and LC conditions stated for the ion trap analysis were used for the triple-quadruple comparison. The instrument was optimized for detecting product ions from the [M + H - H2O]+ ion of dihydroxyvitamin D3 at m/z 399. The transport of m/z 399 ions was done at a cone voltage of 30 V, with optimal CID of the m/z 399 ions accomplished at a collision energy of 25 eV with argon collision gas. The triple quadruple Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

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Figure 2. Comparison of the MS/MS sensitivity for the analysis of a 2 ng/µL solution of lincomycin infused at 20 µL/min using the offaxis nebulizer (A) and on-axis nebulizer (B) for the ion trap MS system.

Figure 3. Spray currents measured at atmospheric pressure for 0.2 mL/min flow rates of water into a pneumatic nebulizer (70 psi of N2, 3 kV). The currents were measured 2.5 cm from the nebulizer tip at angles ranging from 0 to 150° from the nebulizer axis.

was operated under multiple reaction monitoring (MRM) conditions, monitoring the transitions of m/z 399 to 135 and 399 to 213 with a 100-ms dwell time for each transition. RESULTS AND DISCUSSION Evaluating Off-Axis Nebulization on an Ion Trap MS. Offaxis and on-axis pneumatic spray chambers were constructed to permit direct comparison of the two spraying modes on the ITMS using the same instrumental conditions. Figure 1 shows the schematic of the two configurations evaluated. The initial trial of the off-axis nebulizer showed immediate improvement in sensitivity for the infusion of lincomycin into the ITMS (Figure 2). The comparison of the product ion spectra for m/z 407 of lincomycin (infused at 2 ng/µL at 20 µL/min) showed a factor of 6 improvement for the off-axis nebulizer relative to the on-axis nebulizer. This unexpected increase deserved further investigation to understand this gain. The most likely hypothesis was that the off-axis sprayer reduced the number of charged residues that entered and were stored in the ion trap, enabling accumulation of the analyte. This is consistent with the decrease in spray currents as the angle of the nebulizer to capillary entrance is increased (Figure 3). Other explanations include the reduction of charged species at the sampling orifice (capillary) minimizing space charge rejection of the analyte ions and the decrease in the ion trap dampening motion trapping from an increase of gas pressure from solvent droplet evaporation. 1444 Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

Space Charging at Sampling Orifice. To determine whether any of these hypotheses were valid, several experiments were performed. First, the off-axis and on-axis results on the ion trap were compared to an identical experiment on a single quadrupole. The relative ratios for the signal measured off-axis to on-axis was 1.1 on the quadrupole, not the 6-7 observed on the ion trap. This comparison indicated that the improvements in signal intensity obtained on the trap were not due to the reduction of space charge at the sampling orifice in the electrospray transport region (since they are the same in both the quadrupole and ion trap). The capillary current on the electrospray interface was only slightly reduced from 6 × 10-7 to 5 × 10-7A when the nebulizer was switched from on-axis to off-axis. Repeating the experiment on the single quadrupole at different flow rates, the relative signal enhancement for off-axis relative to on-axis nebulization was 1.1, 1.6, and 2.1 at flow rates of 20, 200, and 800 µL/min, respectively. The capillary current ratio between off-axis to on-axis operation showed a slight decrease from 0.8, 0.7, and 0.6 with respect to increasing flow rate. It appears that at higher flow rates there is only a slight improvement attributed to analyte sampling in the API interface due to the reduction of charged species around the capillary entrance from off-axis nebulization. Increase in Solvent Vapor in Ion Trap. Another possible explanation is that for on-axis nebulization the droplets enter the capillary and evaporate throughout the stages in pumping in the system, resulting an increase pressure in the mass analyzer. This could have a detrimental effect on the ion trap sensitivity since solvent gas is less ideal for dampening ion motion compared to helium. When spraying off-axis, only a fraction of the solvent droplets enters the system. To determine the significance of this explanation, the difference in gas composition in the ion trap for on-axis vs off-axis nebulization must be measured. Next, the extent of the increase in solvent gas pressure has on the signal level must be determined. It seems apparent that the solvent contribution would be small since the pressure gauge reading did not vary among on-axis, offaxis, or no solvent flow conditions. However, due to the high pumping speeds used in the vacuum system (520 L/S and 170 L/S turbo pumps), measuring the pump exhaust volume may prove more informative than measuring pressure. The exhaust volume measured at stage 3 (ion guide pressure) was 17.1 ( 0.3 mL/min for no liquid flow, 17.1 ( 0.4 mL/min for off-axis spraying at 0.8 mL/min, and 20.0 ( 0.7 mL/min for on-axis spraying at 0.8 mL/min water (flows measured were in the absence of helium). Stage 4 (ion trap) flows were less than 1 mL/min and could not be accurately measured using the flowmeter. On the basis of these flow measurements, there is an 18% contribution of solvent gas when on-axis nebulization is used. Now the question is, does this gas increase account for the signal difference between on-axis and off-axis nebulization? In normal operation of the ion trap, the pressure measures with helium, nitrogen drying gas, and solvent are at 2 × 10-4 Torr. The partial pressure from helium is 13.2 × 10-5 Torr, and the partial pressure from nitrogen and solvent gas is 6.8 × 10-5 Torr. Accounting for the low response factor of helium relative to nitrogen (response factor of N2 and solvent gas should be close to 1) of ∼0.14, the partial pressure of helium is a minimum of 94.3 × 10-5 Torr. In actuality, the pressure may be 2-3 times

Figure 5. Ion trap end cap current and signal intensity from m/z 407 of lincomycin introduced at 200 mL/min (2 ng/µL × 20 µL injection) from on-axis nebulization as the nitrogen drying gas temperature is increased from 50 to 350 °C. Figure 4. Comparison of the [M + H]+ ion current for lincomycin and end cap current (9), representing the total charged species entering the ion trap, for using the off-axis (A) and on-axis nebulizers (B).

higher since helium is introduced directly into the closed trap. Nevertheless, the nitrogen and solvent account for at most 6.7% of the gas composition under off-axis spraying. For on-axis spraying at 0.8 mL/min, the exhaust flow rate increased by 18%. Assuming that this increase is only from solvent, the gas composition for solvent and nitrogen is increased to 7.9%. This small increase of 1.2% was found to have virtually no effect on signal intensity. The effect of the slight increase in solvent vapor on signal intensity was determined by adding a leak value to stage 3 of the API systems to determine how the increase in water vapor would affect signal level in off-axis spraying. When the leak value was opened to increase the exhaust pump flow rate on stage 3 from 17.1 to 20.8 mL/min (remember on-axis nebulization increased the exhaust flow rate to 20.0 mL/min), there was less than a 30% decrease in signal intensity for the [M + H]+ ion of lincomycin. Charges Residue Effects on Ion Trap Performance. It seems evident that the improved sensitivity observed for off-axis spraying into an ion trap is not due to improved sampling throughout the capillary in the API interface or due to pressure differences caused by solvent evaporation in the vacuum system, rather, the differences observed must be due to the differences in the mass analyzer (ion trap vs quadrupole). To verify this conclusion, the end cap current and signal at m/z 407 for lincomycin were measured at 20, 80, 200, and 800 µL/min for both off-axis and on-axis operation (Figure 4). Figure 4A shows that, when spraying off-axis, the measured signal intensity for m/z 407 is nearly constant and (1.5 × 106 abundance) the end cap currents range from 5 to 10 pA over the four flow rates evaluated (end cap current measures the total of analyte and charged residues can enter the ion trap). In contrast, on-axis spraying resulted in a major decrease in signal level (2.5 × 105 to 0 abundance) and a major increase in end cap current from 30 to 700 pA as flow rate increased. Furthermore, the ratio of end cap currents between on-axis and off-axis increased to 5, 10, 41, and 70 as the flow rate increased from 20, 80, 200, and 800 µL/min. The observation that the m/z 407 ion current decreases while the current detected on the end caps increases can be attributed to other charged species that enter the trap. These charged species are not low-mass

solvent ions since the rf ion guide was used to filter ions below m/z 300. Furthermore, the full-scan mass spectrum indicated that greater than 98% of the ion current was at m/z 407 for lincomycin. Therefore, these charged species must be high-molecular-weight charged residues and charged solvent droplets (m/z >650). Further evidence that supports the increased introduction of charged solvent droplets into the trap under on-axis operation is shown in Figure 5. As the drying gas temperature is increased the end cap current decreases, indicative of lower quantities of charged droplets entering the ion trap due to more effective desolution. Also as the temperature is increased the MS signal m/z 407 of lincomycin is increased, consistent with the decrease in charged residues and improved ion evaporation of the [M + H]+ ion for lincomycin. However, desolvation inside the trap after accumulation of ions and charged residues by adding hold times of up to 1 s prior to CID and the scan offered little improvement in sensitivity. Probably, the lack of heat in the trap or the lack of dry helium prevented effective desolvation within the ion trap. Alternatively, the removal of charged residues after space charge limits will not improve the signal for lincomycin. The molecular weight of these charged residues must far exceed the molecular weight of the analyte being measured. Increasing the gating voltage to +200 V in an attempt to stop the charged residues only resulted in a 5-6% reduction of ion current on the ion trap end cap during on-axis nebulization. (NOTE: 40 V is sufficient to prevent the introduction of the [M + H]+ ion of gramicidin (m/z 1142) into the ion trap.) The use of FNF waveforms during ion accumulation also resulted in little change in signal level, indicating that it was ineffective for removing charged particles. To better understand the magnitude of effect from charged residues and why off-axis nebulization reduces their introduction into the trap, some estimates on droplet size and molecular weight are postulated. A typical nebulizer operating at 1 mL/min (H2O) will produce on average 2-µm-diameter droplets, resulting in the formation of ∼3 × 1010 droplets in 1 min. The molecular weight of the water droplet would be 3.6 × 1014 [18(6.02 × 1023/3 × 1010)], which would represent the worst-case scenario. While droplets of this size could be in the ion trap, more realistically, the size of the droplets that would be found inside the ion trap are on the order of 10 nm in a radius or smaller.3,5 This is more Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

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Table 1. Comparison of Precision for Determination of Dihydroxyvitamin D3 in Plasma on the Ion Trap and Triple-Quadrupole MS % rel std dev instrument configuration

3 ng/mL

30 ng/mL

ion trap LC/MS/MS on-axis nebulization ion trap LC/MS/MS off-axis nebulization triple quadrupoe LC/MS/MS (MRM)

nda 7.1 6.9

14.1 5.6 1.8

a

nd, not detected.

Figure 6. LC/electrospray ion trap analysis, using the off-axis nebulizer, of a 30 ng/mL level of dihydroxyvitamin D3 in plasma. The retention time for dihydroxyvitamin D3 is 1.9 min. (A) Full-scan ion trap analysis. (B) LC/MS/MS analysis monitoring the transitions of m/z 399-381 (C). Ion trap LC/MS/MS using an on-axis nebulizer for the determination of a 30 ng/mL solvent standard.

Figure 7. Comparison of the off-axis and on-axis nebulizers for repetitive analysis for 50 ng/mL level of dihydroxyvitamin D3 in 60 plasma extracts. The plot was generated by monitoring the transition from m/z 399 to 381 in the ion trap.

realistic of the size of droplets that yield ions from ion evaporation. A water droplet of this size would have a molecular weight of 4.5 × 107. From this brief analysis, it is apparent that there are numerous charged residues and the molecular weight of the charged residues can far exceed the molecular weight of a small molecule (e.g., MW 1000) by 4-11 orders in magnitude. This large molecular weight difference is consistent with the reduction of sampling these species off-axis compared to on-axis due to their momentum from nebulization. The force directing the ions into the API-MS is from viscous gas drag of nitrogen drying gas; therefore, increasing the nebulization angle from the sampling orifice reduces the extent the large charge residues are sampled (the charges residue show limited changed in direction from an initial nebulization angle) but has virtually no effect on the sampling of smaller analyte ions. Analysis of Dihydroxyvitamin D3 in Plasma Using the OffAxis Nebulizer on the Ion Trap MS. The analysis of dihydroxyvitamin D3 in plasma was used to evaluate the improvement in performance of the electrospray ITMS system with an off-axis nebulizer relative to the on-axis nebulizer. The separation conditions were chosen to offer maximum sample throughput, while providing a near worst case for interferences from matrix ions and charged particles. Figure 6A shows the total ion current (m/z 100-450) chromatogram for the analysis of dihydroxyvitamin D3 in plasma (off-axis spray). Figure 6B shows the signal from offaxis spraying for dihydroxyvitamin D3 spiked in plasma at 30 ng/

mL. In comparison, the signal for on-axis nebulization of a solvent standard at the 30 ng/mL level is barely detected (Figure 6C). Besides offering improved sensitivity, off-axis nebulization improved the precision of the analysis (Table 1) on the ion trap. The ion trap MS/MS spectra for the [M + H - H2O]+ ion of dihydroxyvitamin D3 exhibited sequential losses of water and some structurally relevant fragments at m/z 254, 265, 278, and 291. The loss of H2O at m/z 381 was the transition chosen for monitoring. Ruggedness of Off-Axis Nebulization for Ion Trap Determination of Dihydroxyvitamin D3. Another benefit of the offaxis relative to on-axis nebulization was the ability to handle a large number of plasma samples with virtually no loss in signal level. Figure 7 compares the signal response for a constant level of dihydroxyvitamin D3 between the off-axis and on-axis nebulizer for the analysis of 60 plasma extracts. The off-axis results exhibited a CV less than 10% during the analysis of the 60 samples while the results with the on-axis nebulizer showed a loss by a factor of 3-4 in signal level at the completion of the 60 samples. The same trend would be observed with quadrupole mass analyzers using an off-axis nebulization. The loss in signal level for on-axis nebulization can be attributed to the physical buildup of matrix components on the end plate and capillary orifice, reducing the sampling efficiency of the ions. Off-axis nebulization resulted in much less residue buildup on the end plates or sampling orifice of the API interface. The majority of the matrix components went in a straight line toward the side of the atmospheric chamber

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D3. Figure 8 shows the LC/MS/MS ion chromatograms for the determination for 3 ng/mL level of dihydroxyvitamin D3 in plasma by the off-axis nebulizer on the ion trap and the triple quadrupole. The sensitivity of the two techniques were similar, with the triple quadrupole showing slightly less than a factor of 2 better S/N compared to the ion trap. The relative standard deviation for the measurement of dihydroxyvitamin D3 was noticeably better at 30 ng/mL by the triple quadrupole compared to the ion trap, but the trap and triple showed similar precision at 3 ng/mL (Table 1). It appears that with the reduction of the charged background species that enter the ion trap, the precision of the ion trap can match those of a triple quadrupoles. Off-axis nebulization on the triple quadrupole should improve detection limits by a factor of 2-3 and may improve the precision relative to an ion trap, based upon improvements observed between off-axis and on-axis API operation on a quadrupole. Figure 8. LC/MS/MS determination for 3 ng/mL dihydroxyvitamin D3 in plasma using (A) off-axis nebulizer ion trap MS system monitoring the CID transition of m/z 399-381 and (B) triplequadrupole system monitoring the CID transition of m/z 399-135.

rather than the sampling orifice as was the case when spraying on-axis. Comparison of Dihydroxyvitamin D3 Determination by Triple-Quadrupole LC/MS/MS. The determination of dihydroxyvitamin D3 in plasma for the off-axis nebulizer ion trap MS system was compared to the determination of identical dihydroxyvitamin D3 plasma extracts using a triple quadrupole with on-axis nebulization. This comparison will permit the evaluation of the off-axis ion trap system, relative to the accepted and proven triplequadrupole LC/MS/MS technique. The triple-quadrupole CID mass spectra showed more extensive fragmentation even with a low collision energy of 25 eV. The triple quadrupole was used to monitor the transitions of m/z 399 to 135 and 399 to 213 for the MRM determination of dihydroxyvitamin D3 in plasma compared to extracting the desired product ions from the full-scan spectrum acquired in the ion trap. Other than monitoring different MRM transitions, the LC conditions were the same for both instruments and the individual mass analyzers were optimized for the formation and CID of the parent ion at m/z 399 ions from dihydroxyvitamin

CONCLUSIONS Charged species formed in the electrospray can drastically affect the performance of the ion trap. Off-axis nebulization reduces the introduction of these charged species into the trap by 1-2 orders in magnitude relative to the on-axis nebulizer. The largest reduction in ion currents from charged species with the off-axis nebulizer is observed when operating at higher flow rates. The off-axis nebulizer exhibits better sensitivity and precision compared to using the on-axis nebulizer. Using the off-axis nebulizer, dihydroxyvitamin D3 could be measured from a plasma extract down to 1 ng/mL with relative standard deviation of less than 8%. The LC/MS/MS sensitivity and precision obtained for dihydroxyvitamin D3 in plasma using the off-axis nebulizer on the ion trap match closely the determinations made on a triplequadrupole MS. ACKNOWLEDGMENT This work was supported by Hoffmann La Roche and FDA Cooperative Agreement FD-U-000589. The triple-quadrupole analyses were performed at the N.C. State University Mass Spectrometry Facility, Raleigh, NC. Received for review February 8, 1999.

September

4,

1998.

Accepted

AC980995S

Analytical Chemistry, Vol. 71, No. 7, April 1, 1999

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