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Separation of Cisplatin and Its Hydrolysis Products Using Electrospray Ionization High-Field Asymmetric Waveform Ion Mobility Spectrometry Coupled with Ion Trap Mass Spectrometry Meng Cui, Luyi Ding,† and Zolta´n Mester*
Institute for National Measurement Standards, National Research Council Canada, Ontario, Canada, K1A 0R6
Cisplatin and its mono- and dihydrated complexes have been separated using a high-field asymmetric waveform ion mobility spectrometry (FAIMS) analyzer interfaced with electrospray ionization (ESI) and ion trap mass spectrometry (ITMS). The addition of helium to the nitrogen curtain/carrier gas in the FAIMS device improved both the sensitivity and selectivity of the electrospray analysis. Introduction of a three-component mixture as curtain/carrier gas, nitrogen, helium, and carbon dioxide, resulted in further improvements to sensitivity. Compared with conventional ESI-MS, the background chemical noise in the ESI-FAIMS-ITMS spectrum was dramatically reduced, resulting in over 30-fold improvement in the signal-to-noise ratio for cisplatin. Analytical results were linear over the concentration range 10-200 ng/mL for intact cisplatin with a corresponding detection limit determined of 0.7 ng/mL with no derivatization or chromatographic separation prior to analysis. Cisplatin is a principal chemotherapeutic drug for cancer treatment. Despite causing severe side effects, it is the preferred treatment for a variety of solid tumors, such as testicular and ovarian cancers, and is also used for treating bladder, cervical, head and neck, esophagean, and small cell lung cancer. However, the modes of action and toxicity of cisplatin are not well understood. It has been suggested that cisplatin and its hydrated species may be responsible for both its antitumor and toxic effects.1-3 The investigation of this premise requires the development of simple, sensitive techniques for the detection of cisplatin and its mono- and dihydrated complexes. Conventional methods for the determination of platinum include atomic absorption spectrophotometry, inductively coupled plasma (ICP) atomic emission * Corresponding author. Tel: (613) 993 5008. Fax: (613) 993 2451. E-mail:
[email protected]. † Current address: Environmental Technology Center, Environment Canada, Ottawa, ON, Canada K1A 0H3. (1) Johnson, N. P.; Hoeschele, J. D.; Rahn, R. O. Chem. Biol. Interact. 1980, 30, 151-169. (2) Barcroft, D. P.; Lepre, C. A.; Lippard, S. J. J. Am. Chem. Soc. 1990, 112, 6860-6871. (3) Daley-Yates, P. T.; McBrien, D. C. H. Biochem. Pharmocol. 1984, 33, 30633070. 10.1021/ac0344182 CCC: $25.00 Published 2003 Am. Chem. Soc. Published on Web 09/16/2003
spectrometry, electrochemical detection, and ICPMS.4-6 However, these techniques only measure platinum content and cannot differentiate intact drug and the active “aquo” species. Methods for the selective determination of platinum species generally resort to hyphenated systems, i.e., liquid chromatographic (LC) separation followed by either on-line or off-line element-specific detection. They cannot provide definitive structural information, however. Such information can be obtained by using 195Pt, 15N, or 1H NMR spectrometry,7 but this requires milligram quantities of material. Currently, the combination of the resolving power of LC with the sensitivity and selectivity of ESI-MS is a promising analytical technique for the determination of platinum-based drugs.8-12 However, when selecting a mobile phase for LC-MS experiments, the reaction chemistry of cisplatin and its hydrolysis products must be considered in order to avoid ambiguities in interpretation of the results.3,13-15 These platinum species react strongly with any nucleophilic compound, such as phosphate, acetonitrile, acetate, and amminonium acetate, which are commonly used in LC mobile phases. Furthermore, this technique is still compromised by the intense matrix-related chemical noise usually associated with ESI from liquid chromatography. An alternative approach is the separation of ions in the gas phase using a technique known as high-field asymmetric waveform ion mobility spectrometry (FAIMS).16-23 The separation of ions (4) Aggorwal, K. S.; Gemma, N. W.; Kinter, M.; Nicloson, J.; Sipe, J. R.; Herold, D. A. Anal. Biochem. 1993, 210, 113-118. (5) Barefoot, R. R. J. Chromatogr., B 2001, 751, 205-211. (6) Premstaller, A.; Ongania, K.; Huber, C. G. Rapid Commun. Mass Spectrum. 2001, 15, 1045-1052. (7) Hahn, M.; Kleine, M.; Sheldrick, S. W. J. Biol. Inorg. Chem. 2001, 6, 556566. (8) Ehrsson, H. C.; Wallin, I. B.; Andersson, A. S.; Edlund, P. O. Anal. Chem. 1995, 67, 3608-3611. (9) Heudi, O.; Cailleus, A.; Allian, P. J. Inorg. Biochem. 1998, 71, 61-69. (10) Oe, T.; Tian, Y.; O’Dwyer, P. J.; Roberts, D. W.; Malone, M. D.; Bailey, C. J.; Blair, I. A. Anal. Chem. 2002, 74, 591-599. (11) Smith, C. J.; Wilson, I. D.; Abou-Shakra, F.; Payne, R.; Parry, T. C.; Sinclair, P.; Roberts, D. W. Anal. Chem. 2003, 75, 1463-1469. (12) Cui, M.; Mester, Z. Rapid Commun. Mass Spectrom. 2003, 17, 1517-1527. (13) Andersson, A.; Fagergerg, J.; Lewensohn, R.; Ehrsson, H. J. Pharm. Sci. 1996, 85, 824-827. (14) Zhao, Z.; Tepperman, K.; Dorsey, J. G.; Elder, R. C. J. Chromatogr. Biomed. Appl. 1993, 615, 83-89. (15) Heudi, O.; Caillieux, A.; Allain, P. Chromatographia 1997, 44, 19-24. (16) Buryakov, I.; Krylov, E.; Nazarov, E.; Rasulev, U. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143-148. (17) Carnahan, B.; Tarassov, A. U.S. Patent 5 420 424, 1995.
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Figure 1. Schematic of the ESI-FAIMS system coupled to a LCQ Deca ion trap mass spectrometer.
by FAIMS is based on compound-dependent differences in ion mobility under the influence of high electric fields (Kh) relative to low electric fields (K). The operating principles of FAIMS were first described for parallel plate geometry by Buryakov et al.16 Later, Carnahan and Tarassov17 improved the design by introducing concentric cylinders to replace the parallel plates. Guevremont et al.18-23 pioneered the use of FAIMS coupled with ESI and quadrupole or TOF mass spectrometers for the separation and focusing of ions. FAIMS acts as an ion filter where a mixture of ions produced by an ESI source is presented to the inlet of the FAIMS analyzer, but only selected ions are transmitted into the mass spectrometer in a continuous fashion. Experimentally, the difference in the ratio of Kh/K is reflected in the value of a compensation voltage (CV) at which one type of ion is selectively transmitted through the device. FAIMS operates at atmospheric pressure and at room temperature. ESI-FAIMS-MS applications include the analysis of complex mixtures, such as haloacetic acids in drinking water,24,25 toxic microcystins,26 peptide fragments from tryptic digestion of proteins,27-30 protein conformers,31-33 and drugs in human urine.34,35 (18) Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszczyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094-4104. (19) Guevremont, R.; Purves, R. W. Rev. Sci. Instrum. 1999, 70, 1370-1383. (20) Purves, R. W.; Guevremont, R. Anal. Chem. 1999, 71, 2346-2357. (21) Guevremont, R.; Purves, R. W.; Barnett, D. A.; Ding, L. Y. Int. J. Mass Spectrom. 1999, 193, 45-56. (22) Guevremont, R.; Purves, R. W.; Barnett, D. A.; Viehland, L. A. J. Chem. Phys. 2001, 14, 10270-10277. (23) Guevremont, R.; Ding, L. Y.; Ells, B.; Barnett, D. A.; Purves, R. W. J. Am. Soc. Mass Spectrom. 2001, 12, 1320-1330. (24) Ells, B.; Barnett, D. A.; Purves, R. W.; Guevremont, R. Anal. Chem. 2000, 72, 4555-4559. (25) Ells, B.; Barnett, D. A.; Froese, K.; Purves, R. W.; Hrudey, S. E.; Guevremont, R. Anal. Chem. 1999, 71, 4747-4752. (26) Ells, B.; Froese, K.; Hrudey, S. E.; Purves, R. W.; Guevremont, R.; Barnett, D. A. Rapid Commun. Mass Spectrom. 2000, 14, 1538-1542. (27) Barnett, D. A.; Guevremont, R.; Purves, R. W. Appl. Spectrosc. 1999, 53, 1367-1374. (28) Barnett, D. A.; Ells, B.; Guevremont, R.; Purves, R. W. J. Am. Soc. Mass Spectrom. 2002, 13, 1282-1291. (29) Barnett, D. A.; Ding, L. Y.; Ells, B.; Purves, R. W.; Guevremont, R. Rapid Commun. Mass Spectrom. 2002, 16, 676-680. (30) Guevremont, R.; Barnett, D. A.; Purves, R. W.; Vandermey, J. Anal. Chem. 2000, 72, 4577-4584. (31) Purves, R. W.; Barnett, D. A.; Guevremont, R. Int. J. Mass Spectrom. 2000, 197, 163-177. (32) Purves, R. W.; Barnett, D. A.; Ells, B.; Guevremont, R. J. Am. Soc. Mass Spectrom. 2000, 11, 738-745.
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In this study, the FAIMS device, a prototype Selectra (Ionalytics, Ottawa, Canada) was interfaced to a LCQ Deca ITMS (Thermo Finnigan, San Jose, CA) for the first time. This ESIFAIMS-ITMS instrument was used to separate cisplatin and its hydrated species by selectively transmitting them through FAIMS followed by determination by mass spectrometry without derivatization or chromatographic separation. Dramatic reductions in background chemical noise and analysis time have been achieved. EXPERIMENTAL SECTION Apparatus. All MS experiments were performed on a Finnigan LCQ Deca mass spectrometer (Thermo Finnigan). ESI-MS. The ESI-MS experiment used a commercial ESI source optimized to produce the most intense ions. Nitrogen was used as sheath gas (100 psi) at a flow rate of 20 arbitrary units. An electrospray voltage of 4.5 kV and a capillary temperature of 200 °C were used. Ultrahigh-purity helium was used as the buffer gas. Samples were infused at 3 µL/min. ESI-FAIMS-MS. The ESI-FAIMS-MS experiments were performed by removing the commercial ESI source and mounting the FAIMS device (Ionalytics Corp.) to the metal capillary of the LCQ mass spectrometer (shown in Figure 1). The FAIMS device used in this study has been described in detail elsewhere.21,30,32 Briefly, it consists of two concentric stainless steel cylinders with the inner surface of the end of the outer cylinder machined to a cancave spherical shape (20-mm i.d.). The outer surface of the end of the inner cylinder (16-mm o.d.) was machined to a hemispherical surface. The FAIMS analyzer region (the space between the two concentric cylinders) was kept at a constant width of 2 mm. The entire device was mounted inside a polyether etherketone (PEEK) housing and operated at atmospheric pressure and room temperature. A 750-kHz asymmetric waveform was applied to the inner cylinder using a custom-built waveform generator. The magnitude of the high-voltage component of the waveform is referred to as the dispersion voltage and could be varied to a maximum of 4000 (33) Purves, R. W.; Barnett, D. A.; Ells, B.; Guevremont, R. J. Am. Soc. Mass Spectrom. 2001, 12, 894-901. (34) McCooeye, M. A.; Mester, Z.; Ells, B.; Barnett, D. A.; Purves, R. W.; Guevremont, R. Anal. Chem. 2002, 74, 3071-3075. (35) McCooeye, M. A.; Ells, B.; Barnett, D. A.; Purves, R. W.; Guevremont, R. J. Anal. Toxicol. 2001, 25, 81-87.
Table 1. Molecular Ion Structures of Cisplatin and Its Hydrolysis Products and Corresponding Theoretical Isotope Distributions
V. A CV was also applied to the inner cylinder using either a 120-V dc power supply (Agilent, model E3612A) for fixed CV measurements or a function generator (Stanford Research System, model DS345) for scanning CV. The electrospray needle (+4100 V, +55 nA) was positioned at an angle of ∼45° and ∼1 cm from a 2-mm opening in the brass curtain plate (1000 V) of the FAIMS device. The sample solutions were delivered at flow rate of 1 µL/min. Industrial-grade nitrogen, helium, or carbon dioxide (Air Products, Ottawa, ON, Canada) was passed through separate charcoal/molecular sieve filters before being combined in a tee assembly and introduced into the region between the curtain plate and the outer cylinder. The gas was split into two flows: the majority of this gas served as the curtain gas flowing out of the opening in the curtain plate countercurrent to the ESI-generated ions, aiding in their desolvation. A smaller portion of the gas flow carried the ions inward through a 1-mm opening in the outer FAIMS cylinder and along the analyzer region of the FAIMS device. This gas is referred to as the carrier gas. Individual gas flows were adjusted using mass flow controllers (MKS type 179A) interfaced to an MKS type 247A four-channel readout (MKS Instruments, Andover, MA). The curtain/carrier gas consisted of pure nitrogen, a mixture of nitrogen and helium, a mixture of nitrogen and carbon dioxide, or a mixture of all three gases. Total flow rate for all experiments was kept constant at 1.8 L/min. Under the appropriate combination of dispersion voltage and compensation voltage, ions could be selectively transmitted through the FAIMS device and focused to a region in front of the spherical tip of the inner cylinder.19 Identification of the ions separated by FAIMS was achieved using the mass spectrometer. A constant dc bias of +50 V was applied to the FAIMS outer cylinder to enhance the sensitivity of the ESI-FAIMS-MS. Reagents. Cisplatin was purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. HPLC-grade
methanol was purchased from Fisher Scientific Co. (Fair Lawn, NJ). Procedure. A 1 mM (hydrolyzed) stock solution of cisplatin was prepared in water at least 24 h before use and was kept in the dark at room temperature. The stock solution contains 30% of intact cisplatin and its mono- and dihydrated species (Table 1) as described in the literature.12,36 A concentrated standard solution containing 3 µg/mL cisplatin (hydrolyzed) was prepared in methanol from the stock solution and was used to investigate the separation of cisplatin and mono- and dihydrated cisplatin using ESI-FAIMS-MS. Calibration solutions of intact cisplatin at 10, 20, 40, 80, and 200 ng/mL were prepared from the stock solution. Each calibration sample was analyzed four times. The mean value of ion intensity at each calibration level was used to plot the calibration curve. The detection limit of cisplatin was calculated based on three times the standard deviation of the zero addition signal. RESULTS AND DISCUSSION Separation of Platinum Species Ions with FAIMS. Using the appropriate combination of dispersion voltage and compensation voltage, ions can be transmitted separately through FAIMS. The effect of varying dispersion voltage on the compensation voltage spectra of platinum species is illustrated in Figure 2. Each trace represents an ion-selected compensation voltage spectrum collected for a 3 µg/mL (hydrolyzed) cisplatin solution. The spectra were acquired by scanning compensation voltage from -20 to 0 V at different dispersion voltage settings and monitoring the m/z values of the most abundant isotope ion of cisplatin, monohydrated complex, and dihydrated complex, as shown in Table 1. Pure nitrogen was used as the carrier gas in this experiment. (36) Verschraagen M.; van der Born, K.; Zwiers, T. H. U.; van der Vijgh, W. J. F. J. Chromatogr., B 2002, 772, 273-281.
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Figure 2. Effect of dispersion voltage on the ion-selected compensation voltage spectra of cisplatin (CP) and its mono- (MP) and dihydrated (DP) complexes: dispersion voltage, (a) 0, (b) 1000, (c) 2500, (d) 3500, and (e) 4000 V. (For clarity, the intensity of dihydrated complex has been multiplied by 5-fold; the intensity of cisplatin has been multiplied by 10-fold.) Figure 4. Ion-selected compensation voltage spectra of cisplatin (CP) and mono- (MP) and dihydrated (DP) complexes collected for the same solution in Figure 2 at dispersion voltage 4000 V: with carrier gas of (a) nitrogen containing 5% CO2; (b) nitrogen containing 20% CO2. (For clarity, the intensity of dihydrated complex has been multiplied by 5-fold; the intensity of cisplatin has been multiplied by 10-fold.)
Figure 3. Mass spectra acquired at compensation voltage values corresponding to the peak maximums in the ion-selected compensation voltage spectrum (Figure 2e): (a) monohydrated complex (MP) and unknown U1 at compensation voltage -11.6 V; (b) dihydrated complex (DP) and unknown U2 at compensation voltage -14.4 V; (c) cisplatin (CP) at compensation voltage -8.5 V.
In Figure 2, trace a shows the compensation voltage spectra at dispersion voltage 0 V. A small peak near compensation voltage 0 V is observed. In the complete absence of applied ac or dc electric potentials, the ions moving along with the carrier gas inside the FAIMS device will not experience any change in their mobilities. This peak is a consequence of the ion diffusion in the 5850 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003
Figure 5. Ion-selected compensation voltage spectra of cisplatin (CP) and mono- (MP) and dihydrated (DP) complexes collected for the same solution in Figure 2 at dispersion voltage 4000 V: with carrier gas of (a) nitrogen containing 5% He; (b) nitrogen containing 20% He; (c) nitrogen containing 40% He. (For clarity, the intensities of dihydrated complex and unknown U2 have been multiplied by 5-fold; the intensity of cisplatin has been multiplied by 10-fold.)
FAIMS analyzer. Trace b was collected at dispersion voltage 1000 V. Due to application of the high-frequency electric field, the ions start to oscillate, but the field is not high enough to make them
Figure 6. Mass spectra acquired at compensation voltage values corresponding to the peak maximums in the ion-selected compensation voltage spectrum (Figure 5c): (a) monohydrated complex (MP) at compensation voltage -18.1 V; (b) dihydrated complex (DP) at compensation voltage -22.8 V; (c) cisplatin (CP) at compensation voltage -10.4 V.
drift toward either cylinder; the spectrum is virtually similar to that in trace a. At dispersion voltage 2500 V, trace c, the electric field is high enough to make ion mobility Kh deviate from its lowfield value K, and hence change the ratio of Kh/K. Three small peaks can be seen at a nonzero value of compensation voltage, corresponding to three platinum species that are still not separated completely by FAIMS at low dispersion voltage. With increasing dispersion voltage, the changes in ion mobility at high field increase and the peaks in the ion-selected compensation voltage spectra shift to the more negative compensation voltage values, which result in better separation of platinum species. At dispersion voltage 3500 V, these peaks become baseline resolved, as shown in Figure 2d. At dispersion voltage 4000 V, trace e, all the peaks shift to more negative compensation voltage and the compensation voltage differences between ions become even larger. Note that the intensity of ions increases substantially and the total peak areas are over 20-fold larger than those in trace a, due to the atmospheric pressure ion focusing mechanism within the FAIMS analyzer.19 Although there appear to be further potential improvements in sensitivity, dispersion voltage values higher than 4000 V could not be investigated due to the voltage limitation of the existing waveform generator. Separation in FAIMS is a function of compensation voltage. Therefore, a compensation voltage value can be set for continuous ion transmission through the FAIMS to allow for MS investigation. The mass spectra acquired at compensation voltage values corresponding to the peak maximums in the ion-selected com-
Figure 7. Ion-selected compensation voltage spectra of cisplatin (CP) and mono- (MP) and dihydrated (DP) complexes of the same solution in Figure 2 at dispersion voltage 4000 V with carrier gas of (a) nitrogen containing 40% He and 3% CO2; (b) nitrogen containing 40% He and 5% CO2; (c) nitrogen containing 40% He and 8% CO2. (For clarity, the intensities of dihydrated complex and unknown U2 have been multiplied by 5-fold; the intensity of cisplatin has been multiplied by 10-fold.)
pensation voltage spectrum (Figure 2e) are illustrated in Figure 3. Each mass spectrum is the average of 10 scans. At compensation voltage -11.6 V, shown in Figure 3a, monohydrated complex is transmitted through FAIMS. The corresponding Zoomscan and tandem MS data are in agreement with previously published data.12 An unknown U1 (m/z 295-299) is also transmitted through FAIMS at this compensation voltage value. The mass spectrum collected at compensation voltage -14.4 V, Figure 3b, shows the transmission of dihydrated complex along with unknown U2 (m/z 277-281). These two unknowns were also found in our previous experiments.12 The optimal compensation voltage to transmit cisplatin is -8.5 V (Figure 3c). The data indicate that the ion mobility of cisplatin and its hydrolysis products (mono- and dihydrated complex) increase with increasing electric field strength, i.e., Kh/K > 1, and these platinum species have different Kh/K values, which allow them to be separated by FAIMS. At optimum combinations of dispersion voltage and compensation voltage, the FAIMS device behaves like an ion filter, capable of transmitting some fraction of a mixture of ions through FAIMS into the mass spectrometer. Improvement of FAIMS Selectivity and Sensitivity. Recent studies have shown that the carrier gas composition affects the Analytical Chemistry, Vol. 75, No. 21, November 1, 2003
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Figure 8. (a) ESI-MS and (b) ESI-FAIMS-MS spectra at dispersion voltage 4000 V and compensation voltage -10.0 V of 200 ng/mL cisplatin.
Figure 9. Calibration curve for cisplatin at dispersion voltage 4000 V and compensation voltage -10.0 V by ESI-FAIMS-MS with nitrogen carrier gas containing 40% He and 3% CO2.
transmission of ions through the FAIMS analyzer.24,37 For some analytes, binary mixtures of carrier gases have been shown to provide better resolution and ion intensities than either of the pure gases alone. (1) Addition of Carbon Dioxide to the Nitrogen Carrier Gas. The effect of using a binary gas mixture of nitrogen and carbon dioxide for the analysis of platinum species is shown in Figure 4. Figure 4a was collected for the same solution in Figure 2 at dispersion voltage 4000 V with nitrogen carrier gas containing 5% CO2. Mono- and dihydrated complex are transmitted through the FAIMS analyzer at less negative compensation voltage values (-13.4 V for dihydrated complex; -11.3 V for monohydrated complex), and cisplatin shifts to more negative compensation voltage -10.0 V. Addition of 5% CO2 to nitrogen carrier gas results (37) Barnett, D. A.; Ells, B.; Guevremont, R.; Purves, R. W.; Viehland, L. A. J. Am. Soc. Mass Spectrom. 2000, 11, 1125-1133.
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in poorer selectivity but better sensitivity for platinum species when compared with the compensation voltage spectra in pure nitrogen (Figure 2e). Increasing the amount of CO2, causes the ion-selected compensation voltage spectra of the three platinum species to overlap although the intensities are improved (Figure 4b). Therefore, addition of CO2 to the nitrogen carrier gas only enhances the sensitivity of the analysis of the platinum species, not their separation. (2) Addition of Helium to the Nitrogen Carrier Gas. The effect of using a binary gas mixture of nitrogen and helium on the analysis of the platinum mixture is shown in Figure 5. Figure 5a shows ion-selected compensation voltage spectra collected for the same solution as in Figure 2 at dispersion voltage 4000 V using nitrogen carrier gas containing 5% He. To demonstrate the improved resolution, the ion-selected compensation voltage spectra of the most abundant ions of the unknown U1 and U2 at m/z 278 and 297 shown in Figure 3 were included. The addition of 5% He to the carrier gas resulted in these three platinum species being transmitted at more negative compensation voltage values (-12.1 V for monohydrated complex; -15.0 V for dihydrated complex; -8.9 V for cisplatin) shown in Figure 5a, and their intensities increased as well. With the addition of 20% He (Figure 5b), monoand dihydrated complexes are transmitted through FAIMS at more negative compensation voltage values with slightly increased intensity. Little change is observed in the compensation voltage spectra of cisplatin. The addition of 40% He has a significant impact on both compensation voltage values and signal intensities of platinum species (Figure 5c). The optimal compensation voltage for transmission of dihydrated complex increases to the highest compensation voltage -22.8 V in the mixed carrier gas, and signal
intensity increases by a factor of 4.5. The monohydrated complex and cisplatin experienced similar increases in compensation voltage values (-18.1 V for monohydrated complex; -10.4 V for cisplatin), and their intensities are higher by a factor of 3 and 3.3, respectively. The separation of target ions (mono- and dihydrated complex) from the unknown U1 and U2 ions has also improved. The mass spectra acquired at compensation voltage values corresponding to the peak maximums of ion-selected compensation voltage spectra shown in Figure 5c are illustrated in Figure 6. Each spectrum represents the sum of 10 scans. Compared with the mass spectra shown in Figure 3, the mono- and dihydrated complexes have been transmitted through FAIMS separated from each other as well as from their impurities. The addition of helium to nitrogen as a carrier gas provides an effective means to improve both selectivity and sensitivity for platinum species. Previous studies have shown that using a mixture of nitrogen and helium as carrier gas provides better resolution and sensitivity than using either of the pure gases alone, for ions having Kh/K < 1 in nitrogen.35 However, in our studies, the platinum species have Kh/K > 1 in nitrogen, and it is surprising that addition of helium to nitrogen carrier gas causes a dramatic increase in both selectivity and sensitivity for these platinum species. (3) Addition of Helium and Carbon Dioxide to the Nitrogen Carrier Gas. A three-gas system (helium, carbon dioxide, nitrogen) as carrier gas was used for analysis of the same solution as shown in Figure 2. Figure 7a shows ion-selected compensation voltage spectra collected at dispersion voltage 4000 V with nitrogen carrier gas containing 40% He and 3% CO2. All the species have largely shifted to less negative compensation voltage values (-17.0 V for dihydrated complex; -13.3 V for monohydrated complex; -10.0 V for cisplatin). And signal intensities increased by a factor of 2, 2, and 1.8 for mono- and dihydrated complexes and cisplatin, respectively, compared with ion-selected compensation voltage spectra shown in Figure 5c. Increasing the CO2 concentration (Figure 7b,c) increases the signal intensities for these platinum species, but the differences in their compensation voltage values are reduced. Comparison of Mass Spectra Collected with ESI-MS and ESI-FAIMS-MS. The advantage of ESI-FAIMS-MS over conventional ESI-MS is demonstrated in Figure 8. Each mass spectrum represents the average of 2-min scanning over a mass range of 100-1000 amu. The ESI-MS spectrum (Figure 8a) is very complicated, and the target cluster ions (cisplatin) are difficult to differentiate from the intense background attributed to other platinum species in the solution as well as to solvent-related background ions. The signal-to-background ratio (S/B) for m/z 323 is estimated to be ∼1 using conventional ESI-MS. Figure 8b shows an ESI-FAIMS-MS spectrum at dispersion voltage 4000 V and compensation voltage -10.0 V. The mass spectrum is quite simple, and the background ions are dramatically reduced. The S/B level is improved over 30-fold relative to the data shown in Figure 8a. The filtering capability of FAIMS allows the target ions
to be separated from intense background prior to their introduction into the mass spectrometer. Quantitative Analysis. A five-point calibration curve for cisplatin is shown in Figure 9. Each point is calculated from the average peak intensities of four injections with relative standard deviation values of 1.3-4.6% (n ) 4). The FAIMS device was tuned for optimal transmission of the cisplatin ions. The limit of detection (LOD) is based on three times the standard deviation of the zero addition signals, and the LOD for cisplatin is 0.7 ng/mL, which is at least 2 orders of magnitude lower than that achieved with conventional ESI-MS.12 This is comparable to the detection limit of 0.1 ng (for a 100-µL injection, corresponding to a concentration determination limit of 1 ng/mL) achieved by LC-ICPMS.14 The day-to-day variation of the system is less than 10% (n ) 5, 10 ng/ mL sample). CONCLUSIONS It is demonstrated that the use of ESI-FAIMS-MS provides a simple, rapid, and sensitive way to separate and detect cisplatin and its hydrolysis products without chromatographic separation prior to the MS analysis. The data indicate that the cisplatin and its hydrolysis products have different Kh/K values, which allow them to be separated by FAIMS. Species separated at a particular CV are available for MS analysis. Using the appropriate combination of dispersion voltage and compensation voltage, three platinum species can be selectively transmitted through FAIMS. Moreover, the filtering ability of FAIMS results in a dramatic reduction in the background and over 30-fold improvement in signal-to-background ratio. The limit of detection for cisplatin by ESI-FAIMS-ITMS is at least 2 orders magnitude lower over conventional ESI-ITMS. Presented data for cisplatin and its hydrolysis products also provide the information to gain better understanding of FAIMS separation. Preliminary experiments showed the use of an asymmetric waveform with dispersion voltage at 4.0 kV provides better separation for target ions. The type of buffer gas used in FAIMS further improves separation of target ions. Adding helium in pure nitrogen extends the CV range at which platinum ions are detected and provide better separation between platinum species. A three-gas system further improved analytical sensitivity. These parameters should be considered during the method development for drug metabolite analysis. ACKNOWLEDGMENT M.C. is grateful to the NRC and NSERC for financial support in the form of a postdoctoral fellowship. The authors thank Dr. D. A. Barnett (Ionalytics Corp., Ottawa, Canada) for his help in installing FAIMS device.
Received for review April 22, 2003. Accepted August 15, 2003. AC0344182
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