Algorithm for Thorough Background Subtraction of High-Resolution

Mar 2, 2009 - 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534. Nonselective collision-induced dissociation (CID) is a technique for ...
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Anal. Chem. 2009, 81, 2695–2700

Algorithm for Thorough Background Subtraction of High-Resolution LC/MS Data: Application to Obtain Clean Product Ion Spectra from Nonselective Collision-Induced Dissociation Experiments Haiying Zhang,*,† Mary Grubb,† Wei Wu,‡ Jonathan Josephs,† and William G. Humphreys† Biotransformation, and Protein Therapeutics Development, Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534 Nonselective collision-induced dissociation (CID) is a technique for producing fragmentation products for all ions generated in an ion source. It is typical of liquid chromatography/mass spectrometry (LC/MS) analysis of complex samples that matrix-related components may contribute to the resulting product ion spectra and confound the usefulness of this technique for structure interpretation. In this proof-of-principle study, a highresolution LC/MS-based background subtraction algorithm was used to process the nonselective CID data to obtain clean product ion spectra for metabolites in human plasma samples. With buspirone and clozapine metabolites in human plasma as examples, this approach allowed for not only facile detection of metabolites of interest but also generation of their respective product ion spectra that were clean and free of matrix-related interferences. This was demonstrated with both an MSE technique (where E represents collision energy) with a quadrupole timeof-flight (QTOF) instrument and an in-source fragmentation technique with an LTQ Orbitrap instrument. The combined nonselective CID and background subtraction approach should allow for detection and structural interpretation of other types of sample analyses where control samples are obtained. Mass spectrometry (MS) is a powerful tool for detection and identification of unknown molecules. Early mass spectrometry was restricted to the detection and identification of low molecular weight organic compounds. Although early mass spectrometers were capable of only one stage of mass analysis, structural information for molecules was readily obtained within the ion source through electron impact (EI) ionization, in which fragmentation products of compounds were formed via unimolecular dissociation.1 With large databases of EI spectra established and coupled with gas chromatography, this mass spectrometry technology continues to have successful applications in many areas for analysis of low molecular mass, volatile compounds.2,3 * To whom correspondence should be addressed. E-mail: haiying.zhang@ bms.com. Phone: 609-818-3537. † Biotransformation. ‡ Protein Therapeutics Development. (1) Sleno, L.; Volmer, D. A. J. Mass Spectrom. 2004, 39, 1091. 10.1021/ac8027189 CCC: $40.75  2009 American Chemical Society Published on Web 03/02/2009

The introduction of fast atom bombardment (FAB),4 electrospray ionization (ESI),5,6 and matrix-assisted laser desorption ionization (MALDI)7,8 in the 1980s has expanded the application of mass spectrometry to a wide range of compounds including those that are large, polar, and/or thermally labile. These soft ionization techniques produce primarily protonated or deprotonated species in the ion source without much formation of structurally informative fragment ions. Structural information is still available but is obtained in a tandem mass spectrometry (MS/ MS) mode where specific molecular ions formed in the ion source are selected and activated, followed by subsequent analysis of fragmentation products formed. The common ion activation method is collision-induced dissociation (CID) in which a fast projectile ion is dissociated as a result of the interaction with a neutral target gas. Depending on the type of instrument, CID can be triggered by high collision energy at kiloelectronvolts or by low collision energy in the 1-100 eV range. The latter is typically used in quadrupole or ion trap instruments. For example, listdependent product ion scans have been routinely employed with ion trap instruments for obtaining product ion spectra of predicted drug metabolites.9-12 Likewise, precursor ion scans and neutral loss scans have been implemented primarily on triple quadrupole instruments for identifying drug metabolites sharing similar fragmentation patterns, i.e., common product ions or neutral losses.13-19 Although product ion spectra may also be obtained (2) Aebi, B.; Bernhard, W. Chimia 2002, 56, 48. (3) Pasikanti, K. K.; Ho, P. C.; Chan, E. C. Y. J. Chromatogr., B 2008, 871, 202. (4) Barber, M.; Bordoli, R. S.; Sedgwick, R. D. In Soft Ionization Biological Mass Spectrometry; Morris, H. R., Ed.; Heyden: London, 1981; p 137. (5) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451. (6) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671. (7) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53. (8) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yohida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151. (9) Yu, X.; Cui, D.; Davis, M. R. J. Am. Soc. Mass Spectrom. 1999, 10, 175. (10) Gangl, E.; Utkin, I.; Gerber, N.; Vouros, P. J. Chromatogr., A 2002, 974, 91. (11) Lafaye, A.; Junot, C.; Gall, B. R.; Fritsch, P.; Tabet, J. C.; Ezan, E. Rapid Commun. Mass Spectrom. 2003, 17, 2541. (12) Anari, M. R.; Sanchez, R. I.; Bakhitiar, R.; Franklin, R. B.; Baillie, T. A. Anal. Chem. 2004, 76, 823. (13) Jackson, P. J.; Brownsill, R. D.; Taylor, A. R.; Walther, B. J. Mass Spectrom. 1995, 30, 446. (14) Clarke, N. J.; Rindgen, D.; Korfmacher, W. A.; Cox, K. A. Anal. Chem. 2001, 73, 430A.

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using in-source fragmentation techniques that activate all ions in the ion source instead of specific molecular ions,20,21 these nonselective CID techniques have not been widely used. One reason is that fragment ions generated may not be easily assigned to a precursor ion due to the nonspecific nature of the activation. This is often problematic because of the high chemical background typically associated with liquid chromatography/mass spectrometry (LC/MS) analysis of complex samples. Nevertheless, in-source fragmentation capabilities have been available. For example, in some Thermo Scientific instruments, there is the option to use a source CID, in which fragmentation occurs between the skimmer and the first multipole region. The advantage is that the spectra obtained are complementary to MSn spectra and there is no one-third cutoff as with typical ion trap instruments.22 Recently an MSE approach (where E represents collision energy) has been reported for an orthogonal hybrid quadrupole time-of-flight instrument (QTOF).23-26 By alternating collision energy settings applied to the collision cell and by using the nonresolving characteristic of Q1, one can obtain data of mostly intact molecular ions from one scan and data of CID fragmentation products from a following scan. This approach of data collection is similar to in-source fragmentation techniques in terms of obtaining nonselective CID spectra.25 The rationale for using this approach is to push for higher throughput of data acquisition and to leverage the accurate mass quality of the data for postacquisition data mining based on specific metabolite masses, precursor and product ions, and neutral losses.23,24 However, it is difficult to properly assign all product ions for a relevant precursor ion, just as with in-source fragmentation. Efforts have been made to link product ions to the relevant precursor ions based on retention time and mass defects.25 More effective ways are needed to eliminate irrelevant product ions that may confound structural interpretation.26 We have recently reported an algorithm for thorough background subtraction from high-resolution LC/MS data.27,28 The algorithm features two characteristics. First, it surveys all data of a control sample within a specified time window around each analyte scan for subtraction of potential sample matrix-related (15) Kostiainen, R.; Kotiaho, T.; Kuuranne, T.; Auriola, S. J. Mass Spectrom. 2003, 38, 357. (16) Lafaye, A.; Junot, C.; Gall, B. R.; Fritsch, P.; Ezan, E.; Tabet, J. C. J. Mass Spectrom. 2004, 39, 655. (17) Baillie, T. A.; Davis, M. R. Biol. Mass Spectrom. 1993, 22, 319. (18) Xia, Y. Q.; Miller, J. D.; Bakhtiar, R.; Franklin, R. B.; Liu, D. Q. Rapid Commun. Mass Spectrom. 2003, 17, 1137. (19) Liu, D. Q.; Hop, C. E. C. A. J. Pharm. Biomed. Anal. 2005, 37, 1. (20) Meng, C. K.; McEwen, C. N.; Larsen, B. S. Rapid Commun. Mass Spectrom. 1990, 4, 151. (21) Rozman, E.; Galceran, M. T.; Albet, C. Rapid Commun. Mass Spectrom. 1995, 9, 1492. (22) Seta, K.; Isobe, T.; Nakayama, H.; Yamaki, S.; Shinkai, F.; Kanai, M.; Xu, H.; Yamaguchi, M.; Okuyama, T. Kuromatogurafi 1996, 17, 344. (23) Clayton, E.; Bateman, R. H.; Preece, S.; Sinclair, I. Adv. Mass Spectrom. 2001, 15, 403. (24) Wrona, M.; Mauriala, T.; Bateman, K. P.; Mortishire-Smith, R. J.; O’Connor, D. Rapid Commun. Mass Spectrom. 2005, 19, 2597. (25) Plumb, R. S.; Johnson, K. A.; Rainville, P.; Smith, B. W.; Wilson, I. D.; CastroPerez, J. M.; Nicholson, J. K. Rapid Commun. Mass Spectrom. 2006, 20, 1989. (26) Bateman, K. P.; Castro-Perez, J.; Wrona, M.; Shockcor, J. P.; Yu, K.; Oballa, R.; Nicoll-Griffith, D. A. Rapid Commun. Mass Spectrom. 2007, 21, 1485. (27) Zhang, H.; Yang, Y. J. Mass Spectrom. 2008, 43, 1181. (28) Zhang, H.; Ma, L.; He, K.; Zhu, M. J. Mass Spectrom. 2008, 43, 1191.

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components in that scan of the analyte sample. Second, the algorithm is executed based on accurate masses of ions detected in the analyte sample. With these two simple measures, matrixrelated signals in the data of an analyte sample are precisely targeted and thoroughly subtracted. This background subtraction approach was used for in vitro screening of glutathione (GSH)trapped reactive metabolites27 and for in vivo profiling of drug metabolites using troglitazone as an example.28 In this communication, we report proof-of-principle results demonstrating the application of this algorithm to nonselective CID data for eliminating sample matrix-related product ions in complex samples. The purpose is to obtain clean, MS/MS-like product ion spectra to facilitate structural interpretation. Both in-source fragmentation and MSE techniques conducted with high-resolution LC/MS instruments were investigated with drug metabolite samples in human plasma. METHODS AND EXPERIMENTAL DETAILS Chemicals and Materials. Acetonitrile was obtained from J. T. Baker (Philipsburg, NJ). Pooled human liver microsomes were from BD Gentest (San Jose, CA). Human plasma (sodium EDTA) was purchased from Bioreclamation (Hicksville, NY). Buspirone and clozapine were obtained from Sigma-Aldrich (St. Louis, MO). Generation of Human Liver Microsomal Incubation Samples. Human liver microsomal incubations were carried out for model compounds buspirone and clozapine. The incubation conditions were as follows: 10 µM compound, 0.5 mg of microsome protein/mL, 80 mM phosphate buffer (pH 7.4), 2 mM NADPH, 5 mM GSH, 37 °C, and 30 min. The incubations were quenched with an equal volume of acetonitrile, and the suspensions were centrifuged at 4000 rpm for 10 min. Aliquots of the supernatants were either analyzed directly by LC/MS or spiked into human plasma followed by LC/MS analysis. Preparation of Human Plasma Samples. An amount of 300 µL of the supernatant from each of the above incubations was dried under a stream of nitrogen and was dissolved into 100 µL of human plasma (Bioreclamation). After adding 200 µL of methanol for protein precipitation, the suspension was centrifuged at 4000 rpm for 10 min. The supernatants were analyzed directly by LC/MS. High-Performance Liquid Chromatography. HPLC was performed using an Atlantis dC18 column (3 µm particle size, 2.1 mm × 150 mm, Waters, Milford, MA) with a flow rate of 250 µL/ min. The HPLC solvents were water and acetonitrile both containing 0.1% formic acid. The HPLC gradient was initiated at 2% acetonitrile, held for 2 min, and then linearly ramped to 40% acetonitrile in 28 min. After raising the gradient to 95% acetonitrile and holding for 5 min, the gradient was returned to the initial conditions and reequilibrated for 5 min. The sample injection volume was 15 µL. QTOF Mass Spectrometry with Low and High Collision Energy Switching. For obtaining nonselective CID data through low and high collision energy switching in a collision cell (MSE), a Synapt QTOF mass spectrometer (Waters, Manchester, U.K.) equipped with an electrospray interface was used in-line with the HPLC system described above. The QTOF instrument was tuned to 9000 resolution at half-peak height and was calibrated using a sodium iodide solution. The LC/MS data were acquired

with two alternating TOF scanning functions: one at a low collision energy of 6 V; one at a high collision energy of 25 V. The data in centroid format were acquired in the range of m/z 100 to m/z 1000 for low collision energy scans and in the range of m/z 80 to m/z 1000 for high collision energy scans. The capillary voltage was 2.5 kV, and the desolvation temperature was 350 °C. The data were acquired without applying any lock mass correction. Fourier Transform Mass Spectrometry with Source CID. For obtaining nonselective CID data through in-source fragmentation, an LTQ Orbitrap Fourier transform mass spectrometer (FTMS) (Thermo Scientific, Bremen, Germany) equipped with an electrospray interface was used in-line with the HPLC system described above. The mass resolution of the FTMS was set to 7500. The instrument was stabilized in the FT scan mode for at least 10 h prior to the study. Mass calibration was performed on the FTMS prior to the data acquisition. The LC/MS data were acquired using two alternating FT scanning functions: one with the source CID option turned off; one with the source CID option turned on. The source CID voltage was set to 60 V. The data in centroid format were acquired in the range of m/z 100 to m/z 1000 for scans with the source CID option off and in the range of m/z 80 to m/z 1000 for scans with the source CID option on. The source voltage was 3 kV, and the capillary temperature was 400 °C. Background Subtraction. Data from both non-CID and nonselective CID experiments were converted to NetCDF format using file conversion utilities provided by the instrument vendors. The LTQ Orbitrap data were preprocessed with a Slicer utility in RecalOffline (Thermo Scientific) to separate the non-CID and the source CID scans into different data files prior to format conversion. Background subtraction was applied to both the non-CID data and the nonselective CID data. In this proof-of-principle study with human plasma, the clozapine samples were used as controls to provide matrix component coverage for the analysis of the buspirone samples, and the GSH-supplemented buspirone samples were used as controls for the analysis of the GSH-supplemented clozapine samples. The background subtraction algorithm has been described elsewhere.27,28 In brief, the software defined a range of control scans based on a specified time window around an analyte scan (±0.3 min in this study) so that the accurate mass data contained within that window could be considered for matrix ion checking. The dynamic “range of control scans” algorithm was looped throughout the analyte scans in an LC/MS data set. Once the control scans were defined for an analyte scan, the actual subtraction of any ion in the spectrum of the analyte scan was performed by first identifying the same ion in the spectra of the control scans (masses in analyte vs control data were matched as long as they fell within a specified mass tolerance window around the analyte masses, which was set to ±20 ppm in this study). The highest intensity of the identified ion in the spectra of the control scans was then determined and multiplied with a specified scaling factor (2) and was subtracted from that of the ion in the spectrum of the analyte scan. After data processing, the output NetCDF file was converted back to the native data format for viewing.

Figure 1. Base peak ion chromatograms of (a) buspirone metabolites from a human microsomal incubation (data acquired with QTOF) and (b) clozapine metabolites from a human microsomal incubation supplemented with GSH (data acquired with LTQ Orbitrap). The chromatogram in panel b is presented in a mass range of m/z 300 to m/z 1000 to cut off some interference peaks from the incubation matrixes. All other chromatograms shown in this paper are presented with full scan mass ranges.

Figure 2. Base peak ion chromatograms of buspirone metabolites in human plasma (data acquired in MSE experiments with QTOF): (a) original data of low collision energy scans; (b) original data of high collision energy scans (CID data); (c) background-subtracted data of low collision energy scans; (d) background-subtracted data of high collision energy scans.

RESULTS Base peak ion chromatograms of buspirone and clozapine metabolites obtained from incubations with human microsomes are shown in Figure 1. These chromatograms serve as reference profiles representing the metabolites of interest that were spiked into the human plasma samples to be discussed below. These reference profiles allow for objective evaluation of the effect of background subtraction toward elimination of matrix-related components from spiked human plasma samples. Analytical Chemistry, Vol. 81, No. 7, April 1, 2009

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Figure 3. Mass spectra of the buspirone M5 peak at RT 18.9 min: (a) original spectrum from the low collision energy scan; (b) original spectrum from the high collision energy scan (CID spectrum); (c) backgroundsubtracted spectrum from the low collision energy scan; (d) backgroundsubtracted spectrum from the high collision energy scan.

Results from MSE Experiments with QTOF. In Figure 2, chromatograms are displayed showing results of MSE experiments conducted on a QTOF instrument of the buspirone metabolitespiked human plasma samples. In the unprocessed original profiles of both the low collision energy scans and the high collision energy scans, significant plasma matrix components dominate the profiles. In the background-subtracted profiles including the high collision energy scans, buspirone metabolites are revealed as major distinct peaks, and the peak profiles are comparable with the reference profile as shown in Figure 1a. In Figure 3, mass spectra are shown to illustrate the effectiveness of using background subtraction for data obtained in MSE experiments for determination of metabolite species and their product ions. As shown in Figure 3, parts a and b, the original unprocessed MS and CID spectra of buspirone metabolite M5 at RT 18.9 min show significant interference from plasma matrix components. After background subtraction, the protonated species of the metabolite and one of its fragments at m/z 308 are prominent as the only major ions in the MS spectrum from the low collision energy scan (Figure 3c). More interestingly, all product ions of the metabolite are displayed as the only major ions in the CID spectrum from the high collision energy scan (Figure 3d). Table S1 of the Supporting Information summarizes this and other buspirone metabolites detected in the buspirone human plasma sample, with product ions listed that were major ions once revealed in the background-subtracted CID spectra. Results from Source CID Experiments with an LTQ Orbitrap. In a similar fashion, Figure 4 shows chromatographic results of the source CID experiments conducted on an LTQ Orbitrap instrument for the clozapine metabolite-spiked human 2698

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Figure 4. Base peak ion chromatograms of clozapine metabolites in human plasma (data acquired in source CID experiments with an LTQ Orbitrap): (a) original data of “source CID off” scans; (b) original data of “source CID on” scans; (c) background-subtracted data of “source CID off” scans; (d) background-subtracted data of “source CID on” scans.

Figure 5. Mass spectra of the clozapine M1 peak at RT 14.0 min: (a) original spectrum from the “source CID off” scan; (b) original spectrum from the “source CID on” scan; (c) background-subtracted spectrum from the “source CID off” scan; (d) background-subtracted spectrum from the “source CID on” scan.

plasma samples. Figures 5 and 6 display MS and CID spectra from the background-subtracted data, together with the respective

Figure 6. Mass spectra of the clozapine M3 peak at RT 18.6 min: (a) original spectrum from the “source CID off” scan; (b) original spectrum from the “source CID on” scan; (c) background-subtracted spectrum from the “source CID off” scan; (d) background-subtracted spectrum from the “source CID on” scan.

unsubtracted spectra. Again, with background subtraction, the clozapine metabolites in human plasma are the only distinct peaks, and the peak profiles are comparable with the reference profile shown in Figure 1b. Also, all product ions of the metabolites are the only major ions in the background-subtracted CID spectra from the source CID scans (Figures 5d and 6d). Table S2 of the Supporting Information summarizes the clozapine metabolites detected in the human plasma sample, along with their product ions listed that were revealed in the background-subtracted CID spectra. DISCUSSION The ability to eliminate matrix-related species from nonselective CID data is desirable. Even with the separations available with modern liquid chromatography, matrix-related components almost always coelute or elute as background with analytes of interest, especially with complex samples such as human plasma. Although molecular ions of drug metabolites sometimes can be predicted based on knowledge of biotransformation pathways (e.g., the m/z 402 ion of several buspirone metabolites and the m/z 343 ion of clozapine metabolites), significant interfering ions present in the product ion spectra from nonselective CID experiments would severely compromise the ability to elucidate the structure of these metabolites. This is shown in the typical mass spectra in Figures 3b, 5b, and 6b. For example, the presence of m/z 155, m/z 287, and m/z 377 ions in Figure 3b would make it difficult to judge whether these ions are derived from the m/z 418 ion. Likewise, the presence of multiple background ions in the product ion spectra of many of the other metabolites listed in Tables S1 and S2 of the Supporting Information would make it difficult or impossible to assign fragmentation patterns without removal of

the background ions. In this proof-of-principle study, however, we have demonstrated that with proper control samples and highresolution LC/MS data it is possible to use a background subtraction algorithm to precisely and thoroughly remove matrix ions in the data of analyte samples. The product ion spectra in the resulting simplified data are so clean that they are like those obtained from selective MS/MS experiments. The quality of such product ion spectra truly allows for proper fragmentation assignments and facilitates the throughput of LC/MS analysis for complex samples. The fundamental advantage of a nonselective CID experiment is that it obtains product ions of all precursors, whereas a datadependent MS/MS experiment obtains structural information for only a few abundant ions, which in many cases may not even be analyte-related. Therefore, a nonselective CID technique in combination with background subtraction represents an attractive choice relative to data-dependent MS/MS, as structural information may be obtained for all analytes of interest in a complex sample. Aside from the MSE and in-source fragmentation techniques mentioned in this study, other nonselective CID techniques would also benefit from this background subtraction approach to obtain clean product ion spectra, provided that accurate mass LC/MS data are acquired. The fragmentation products generated with the MSE and insource fragmentation techniques in this study (Tables S1 and S2 of the Supporting Information) are typical of low collision energy CID experiments and are comparable with those reported in the literature.29-34 Because of the nonselective nature, related precursor ions of an analyte species may all contribute to the formation of the fragmentation products in a spectrum. For example, both the doubly charged ion m/z 316 and the singly charged ion m/z 632 in Figure 6c would contribute to the formation of the product ions shown in Figure 6d. This is an advantage of the nonselective CID methodology,35 as this would provide more information than could be obtained by simply activating the m/z 632 ion in a selective CID experiment. Another advantage associated with the nonselective CID experiment is that the isotope patterns are intact for product ions, making it easy to determine whether or not they contain certain halogen atoms, e.g., the chlorine-containing ion m/z 243 and the dechlorinated ion m/z 208 in Figure 5d. Some adduct ions of molecular species may survive a nonselective CID process and remain in the resulting CID spectra, e.g., the m/z 686 ion in Figure 6d. For insource fragmentation experiments conducted on the LTQ Orbitrap, a relatively high capillary temperature setting was found necessary to maintain fragmentation efficiency with the LC flow. A potential concern for simultaneous acquisition of product ion spectra in a nonselective mode is that product ions of two closely eluting analytes of interest may not be distinguishable from (29) Kerns, E. H.; Rourick, R. A.; Volk, K. J.; Lee, M. S. J. Chromatogr., B 1997, 698, 133. (30) Zhang, M. Y.; Pace, N.; Kerns, E. H.; Kleintop, T.; Kagan, N.; Sakuma, T. J. Mass Spectrom. 2005, 40, 1017. (31) Zhu, M.; Zhao, W.; Jimenez, H.; Zhang, D.; Yeola, S.; Dai, R.; Vachharajani, N.; Mitroka, J. Drug Metab. Dispos. 2005, 33, 500. (32) Fandino, A. S.; Naegele, E.; Perkins, P. D. J. Mass Spectrom. 2006, 41, 248. (33) Aravagiri, M.; Marder, S. R. J. Pharm. Biomed. Anal. 2001, 26, 301–311. (34) Maggs, J. L.; Williams, D.; Pirmohamed, M.; Park, B. K. J. Pharmacol. Exp. Ther. 1995, 275, 1463. (35) Zhang, Z.; Shah, B. Anal. Chem. 2007, 79, 5723.

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one another. This is a relatively minor concern compared to the issue of interferences with sample matrixes, as in most cases a good LC method is capable of separating most analytes of interest. In addition, with simplified data from background subtraction, one can use the Biller-Biemann algorithm36 in some existing MS software packages to reconstruct product ion spectra for closely eluting analyte peaks. For drug metabolite analysis, we found it helpful to include a third scan function along with the source CID on-and-off scans with the LTQ Orbitrap instrument: a datadependent MS/MS acquisition triggered by the presence of ions of accurate masses of predicted metabolites. This data-dependent scan function may not provide CID spectra for all the metabolites detected, but it provides a complementary data set that can be used to check and resolve any concerns about the nonselective CID data. In summary, we have demonstrated that, with proper controls and a thorough background subtraction algorithm, one can not

only detect analytes of interest in complex matrixes but can also simultaneously obtain clean product ion spectra from nonselective CID experiments. This type of high-resolution LC/MS-based data analysis should be applicable to many types of sample analyses as long as proper control samples can be defined and obtained.

(36) Biller, J. E.; Biemann, K. Anal. Lett. 1974, 7, 515.

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ACKNOWLEDGMENT We thank Xin Wang for developing and implementing the background subtraction algorithm. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review December 22, 2008. Accepted February 9, 2009.