High-Field Asymmetric Waveform Ion Mobility Spectrometry for

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Anal. Chem. 2009, 81, 7839–7843

Technical Notes High-Field Asymmetric Waveform Ion Mobility Spectrometry for Determining the Location of In-Source Collision-Induced Dissociation in Electrospray Ionization Mass Spectrometry Yuan-Qing Xia and Mohammed Jemal* Bioanalytical and Discovery Analytical Sciences, Research & Development, Bristol-Myers Squibb, Princeton, New Jersey 08543 The understanding and control of the in-source collisioninduced dissociation (CID) of analytes is important for the accurate LC-MS/MS quantitation of drugs and metabolites in biological samples. Accordingly, it was of interest to us to establish whether such in-source CID takes place after and/or before the orifice of an electrospray ionization (ESI) mass spectrometer. A high-field asymmetric waveform ion mobility spectrometry (FAIMS) system that is physically located between the sprayer and the orifice of a mass spectrometer can serve as an ion filter to control ions entering the orifice of the mass spectrometer. In such a configuration, FAIMS could conceivably be used to determine if the in-source CID of an analyte occurs after and/or before the mass spectrometer orifice. We demonstrated this capability of FAIMS using ifetroban acylglucuronide metabolite as a model compound. Under the conditions used, the results showed that the in-source CID conversion of the acylglucuronide metabolite to its parent drug ifetroban occurred almost entirely after the orifice of the mass spectrometer, with the conversion upstream of the orifice accounting for only 5.6% of the conversion. Under the circumstance, the term “postorifice CID” rather than “in-source CID” may be more appropriate in describing such a dissociation occurring in the front end of a mass spectrometer. Electrospray ionization (ESI) is widely used in liquid chromatography mass spectrometry (LC-MS) quantitation of drugs, metabolites, and biomarkers in biological samples.1-3 In ESI, a product ion may be generated from the molecular ion of a compound in the source via collision-induced dissociation (CID) depending on the source parameters used, such as declustering or tube lens potential and source temperature. The understanding * Corresponding author. Address: Route 206 & Province Line Road, Princeton, NJ 08543-4000. E-mail: [email protected]. Phone: 609-252-3572. (1) Korfmacher, W. A. Drug Discovery Today 2005, 10, 1357–1367. (2) Jemal, M.; Xia, Y. Q. Curr. Drug Metab. 2006, 7, 491–502. (3) Bakhtiar, R.; Ramos, L.; Tse, F. L. S. J. Liq. Chromatogr. Relat. Technol. 2002, 25, 507–540. 10.1021/ac9012336 CCC: $40.75  2009 American Chemical Society Published on Web 08/19/2009

and control of such in-source CID is important in order to avoid potential interference in the accurate quantitation of a drug since its prodrug and/or its metabolites may undergo in-source CID to generate the drug.4,5 In-source CID is generally presumed to occur after the mass spectrometer orifice, in the skimmer region.6-12 However, it is possible that some in-source CID may also take place before the orifice. Herein, we demonstrate the use of highfield asymmetric waveform ion mobility spectrometry (FAIMS) to determine the amount of the CID conversion taking place before the orifice vis-a`-vis that taking place after the orifice. Applications and detailed discussions of the fundamental principles of FAIMS have been presented in previous publications.13-20 A FAIMS system that is physically located between the ESI sprayer and the orifice can be used as an ion filter to select the ions entering the orifice of the mass spectrometer, thereby discarding the (4) Jemal, M.; Xia, Y. Q. Rapid Commun. Mass Spectrom. 1999, 13, 97–106. (5) Jemal, M.; Ouyang, Z.; Powell, M. L. Rapid Commun. Mass Spectrom. 2002, 16, 1538–1547. (6) Schneider, B. B.; Chen, D. D. Y. Anal. Chem. 2000, 72, 791–789. (7) Chen, H.; Tabei, K.; Siegel, M. M. J. Am. Soc. Mass Spectrom. 2001, 12, 846–852. (8) Li, J.; Wang, Z.; Altman, E. Rapid Commun. Mass Spectrom. 2005, 19, 1305– 1314. (9) Schneider, B. B.; Douglas, D. J.; Chen, D. D. Y. Rapid Commun. Mass Spectrom. 2001, 15, 249–257. (10) Kubwabo, C.; Vais, N.; Benoid, F. M. Rapid Commun. Mass Spectrom. 2005, 19, 597–604. (11) Tuytten, R.; Lemiere, F.; Esmans, E. L.; Herrebout, W. A.; van der Veken, B. J.; Dudley, E.; Newton, R. P.; Witters, E. J. Am. Soc. Mass Spectrom. 2006, 17, 1050–1062. (12) Williams, J. D.; Flanagan, M.; Lopez, L.; Fischer, S.; Miller, L. A. D. J. Chromatogr., A 2003, 1020, 11–26. (13) Guevremont, R. J. Chromatogr., A 2004, 1058, 3–19. (14) Shvartsburg, A. A.; Tang, K.; Smith, R. D. Anal. Chem. 2004, 76, 7366– 7374. (15) Shvartsburg, A. A.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2005, 16, 2–12. (16) Kapron, J.; Jemal, M.; Duncan, G.; Kolakowski, B. M.; Purves, R. Rapid Commun. Mass Spectrom. 2005, 19, 1979. (17) Shvartsburg, A. A.; Li, F.; Tang, K.; Smith, R. D. Anal. Chem. 2006, 78, 3706–3714. (18) Kolakoswski, B. M.; McCooeye, M. A.; Mester, Z. Rapid Commun. Mass Spectrom. 2006, 20, 3319–3329. (19) Wu, S. T.; Xia, Y. Q.; Jemal, M. Rapid Commun. Mass Spectrom. 2007, 21, 3667–3676. (20) Xia, Y. Q.; Wu, S. T.; Jemal, M. Anal. Chem. 2008, 80, 7137–7143.

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Table 1. FAIMS Parameters Used ifetroban ifetroban acylglucuronide dispersion voltage (V) total gas flow (helium/nitrogen, L/min) % helium/nitrogen inner/outer electrode temperatures (°C) out bias voltage (V)

Figure 1. Chemical structures of ifetroban and its acylglucuronide metabolite.

unwanted ions. The ion selection in FAIMS is achieved by applying a compensation voltage (CV) that is specific to the ion of interest. Thus, the molecular ion of a compound can be separated from its CID-generated product ion. Consequently, experiments can be designed using FAIMS to determine if the CID-generated product ion is produced before or after the orifice of the mass spectrometer. Herein, we demonstrate such an approach using an acylglucuronide metabolite as a model compound undergoing in-source CID conversion. EXPERIMENTAL SECTION Materials and Apparatus. Ifetroban and its acylglucuronide metabolite (Ife-Glu) are proprietary compounds from BristolMyers Squibb Company (Princeton, NJ). The chemical structures of the compounds are shown in Figure 1. A Thermo Finnigan TSQ Quantum Ultra mass spectrometer equipped with a heatedelectrospray ionization (HESI) source and FAIMS system (San Jose, CA) was used. The LC system used consisted of two Shimadzu LC-ADVP binary pumps (Columbia, MD) coupled with a Shimadzu SIL-HT autosampler (Columbia, MD). The analytical column, Atlantis dC18 (2.1 × 50 mm, 3 µm), was purchased from Waters (Milford, MA). Formic acid (98%) and HPLC-grade acetonitrile were purchased from EM Science (Gibbstown, NJ). House nitrogen (99.99%) and ultra high purity helium from Airgas (Radnor, PA) were used. Mobile phase A was water and mobile phase B was acetonitrile, both containing 0.1% formic acid. Liquid Chromatography FAIMS Mass Spectrometry. The mass spectrometer was operated in the positive ESI mode. Selected reaction monitoring (SRM) was used for ifetroban (m/z 441 f m/z 423) and Ife-Glu (m/z 617 f m/z 423). Nitrogen was used as the ESI sheath and auxiliary gas and was set to 60 and 50 psi, respectively. The ESI voltage was set at 4000 V. The heated electrospray vaporizer temperature and the capillary temperature were set at 350 and 250 °C, respectively. Other parameters, such as capillary and tube lens offsets, were optimized. Chromatographic data were acquired and processed using Thermo Finnigan Xcalibur 2.0 SR2, which was also used to control FAIMS. The scan time was 0.05 s and the scan width was 0.5 Da for each SRM transition. CV profiles of ifetroban and Ife-Glu were determined separately by conducting postcolumn infusion of the analyte solution (0.5 µM) at 5 µL/min into 50:50 mobile phase A/mobile phase B flowing at 0.4 mL/min into the FAIMS mass spectrometer. The FAIMS parameters used are listed in Table 1. For the determination of the location of the in-source CID of Ife-Glu, the LC system used was optimized to achieve the 7840

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-5000 3.5 40/60 100/80 35

-5000 3.5 40/60 100/80 35

chromatographic separation of Ife-Glu from ifetroban. To this end, the Atlantis dC18 column (2.1 × 50 mm, 3 µm) was used with a gradient elution at flow rate of 0.4 mL/min. The gradient elution was as follows: start at 30% B and hold there for 0.2 min; increase from 30% B to 90% B in 3.3 min and then hold there for 0.5 min; decrease from 90% B to 30% B in 0.1 min and hold there for 0.9 min. The run time was 5 min. The column temperature was 30 °C, and the injection volume was 5 µL. RESULTS AND DISCUSSION Acylglucuronide metabolites such as Ife-Glu have been reported to undergo in-source CID conversion to generate the corresponding parent drugs via the loss of the glucuronide moiety (176 Da).4,5 Thus, Ife-Glu will, under appropriate positive ESI conditions, produce in the source not only its [M + H]+ ion (m/z 617) but also the [M + H]+ ion of the parent drug ifetroban (m/z 441). Consequently, these precursor ions will undergo MS/MS fragmentation in the Q2 of a triple quadrupole mass spectrometer. For the investigation described herein, the disposition of these ions was studied employing the SRM of m/z 617 f m/z 423 for Ife-Glu and m/z 441 f m/z 423 for ifetroban. In order to determine the location of the in-source CID of Ife-Glu, it was important to determine the FAIMS CV profiles of Ife-Glu and ifetroban. As shown in Figure 2, the CV profiles

Figure 2. Compensation voltage profiles of ifetroban and its acylglucuronide metabolite.

Figure 3. LC-FAIMS-SRM chromatograms obtained from the injection of a sample containing only ifetroban acylglucuronide (Ife-Glu) with the CV set at -10.7 V (optimum CV value for Ife-Glu): (a) ifetroban SRM channel (m/z 441 f m/z 423); (b) Ife-Glu SRM channel (m/z 617 f m/z 423).

of the two compounds are adequately resolved with the optimum CV value of -13.7 V for ifetroban and -10.7 V for Ife-Glu. Thus, using a CV setting of -10.7 V with an LC-FAIMSMS system that chromatographically separates ifetroban (2.94 min) from Ife-Glu (2.36 min), a sample containing the two analytes is expected to give response only in the Ife-Glu SRM channel (m/z 617 f m/z 423) at the retention time Ife-Glu, with little-to-no response in the ifetroban SRM channel (m/z 441 f m/z 423) at the retention time of ifetroban. On the other hand, using a CV setting of-13.7 V, the same sample is expected to give response only in the ifetroban SRM channel at the retention time ifetroban, with little-to-no response in the Ife-Glu SRM channel at the retention time of Ife-Glu. This unique selectivity of FAIMS in discriminating a precursor ion from its in-source CID product ion is the basis for the determination of the location of the in-source CID of the precursor ion. As presented below, the location of the in-source CID of Ife-Glu was established by injecting a sample containing only Ife-Glu (with no ifetroban) into the LC-FAIMS-MS system with the CV set at either -10.7 or -13.7 V using chromatographic conditions that separated Ife-Glu from ifetroban. The SRM chromatograms obtained with CV set at -10.7 V are shown in Figure 3. As expected, the Ife-Glu SRM channel (m/z 617 f m/z 423) showed a peak at the retention time of Ife-Glu (2.36 min, Figure 3b) since the CV used allowed the transmission of the m/z 617 ion (protonated Ife-Glu) through the FAIMS system. In contrast, the ifetroban SRM channel (m/z 441 f m/z 423) did not show a peak at the retention time of ifetroban (2.94 min, Figure 3a) since the CV used would prevent the transmission of the m/z 441 ion (protonated ifetroban), even if the injected sample were to contain ifetroban. On the other hand, the ifetroban SRM channel showed a

peak at the retention time of Ife-Glu (2.36 min, Figure 3a). This is attributed to the in-source CID conversion of the Ife-Glu m/z 617 ions to the ifetroban m/z 441 ions. Since the FAIMS system would not allow the transmission of the m/z 441 ions, it is concluded that the m/z 441 ions detected under the CV value of -10.7 V (optimum CV for Ife-Glu) were generated from the Ife-Glu m/z 617 ions that were transmitted through the FAIMS device and the mass spectrometer orifice. The SRM chromatograms obtained with CV set at -13.7 V are shown in Figure 4. The summary of the comparison of the results from Figures 3 and 4 are shown in Table 2. Ideally, the Ife-Glu SRM m/z 617 f m/z 423 channel in Figure 4b was not expected to give a peak at the retention time of Ife-Glu (2.36 min) because the m/z 617 ions were supposed to be filtered away due to the nonoptimal CV of -13.7 V used. However, a SRM peak of m/z 617 f m/z 423 was seen at 2.36 min (Figure 4b) amounting to 4.2% ((4.41 × 104/1.05 × 106) × 100 ) 4.2%) of the corresponding peak seen in Figure 3b. This is attributed to a minor “leakage” of the m/z 617 ions through the FAIMS system under the conditions used. The ifetroban SRM m/z 441 f m/z 423 channel also showed a peak at the same 2.36 min retention time (Figure 4a). In the absence of the minor leakage of the m/z 617 ions through the FAIMS system, the 2.36 min ifetroban SRM channel peak observed in Figure 4a (7.59 × 104) would have been attributed to the insource conversion of Ife-Glu entirely occurring upstream of the FAIMS system, probably in the vicinity of the ESI sprayer. The ifetroban peak response arising from the leakage of the m/z 617 ions is calculated to be 3.26 × 104 ((4.41 × 104) × 0.74 ) 3.26 × 104). Thus, the corrected ifetroban response, which is attributed to the conversion of Analytical Chemistry, Vol. 81, No. 18, September 15, 2009

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Figure 4. LC-FAIMS-SRM chromatograms obtained from the injection of a sample containing only ifetroban acylglucuronide (Ife-Glu) with the CV set at -13.7 V (optimum CV value for ifetroban): (a) ifetroban SRM channel (m/z 441 f m/z 423); (b) Ife-Glu SRM channel (m/z 617 f m/z 423).

Table 2. Summary of the Results Obtained from the Injection of a Sample Containing Only Ifetroban Acylglucuronide (Ife-Glu) into LC-FAIMS-MS at a Compensation Voltage (CV) Setting of -10.7 or -13.7 Va observed peak area at retention time of 2.36 min

calculated peak area for ifetroban at retention time of 2.36 min

CID conversion CID conversion ifetroban Ife-Glu occurring after occurring before compensation SRM channel SRM channel FAIMS-MS orifice FAIMS-MS orifice CID conversion voltage used (V) (m/z 441 f m/z 423) (m/z 617 f m/z 423) (m/z 441 f m/z 423) (m/z 441 f m/z 423) (%) -10.7 (optimal for Ife-Glu)

7.75 × 105b

1.05 × 106b

7.75 × 105

NAd

74e

-13.7 (optimal for ifetroban)

7.59 × 104c

4.41 × 104c

3.26 × 104f

4.33 × 104g

5.6h

comment The ifetroban response (7.75 × 105) indicates major CID occurring after the FAIMS-MS orifice region. The calculated ifetroban response (4.33 × 104) indicates minor CID occurring before the FAIMS-MS orifice region.

a The corresponding SRM chromatograms are shown in Figures 3 and 4. b Experimentally obtained peak area values as shown in Figure 3. Experimentally obtained peak area values as shown in Figure 4. d NA: not applicable. e This represents the degree of CID conversion occurring after the FAIMS-MS orifice, calculated as (7.75 × 105/1.05 × 106) × 100 ) 74%. f This is the ifetroban SRM peak response expected from the conversion of the Ife-Glu ions responsible for the measured Ife-Glu peak response of 4.41 × 104, calculated as (4.41 × 104) × 74% ) 3.26 × 104. g This represents the estimated ifetroban response due to the conversion of the Ife-Glu ions before the FAIMS-MS orifice, calculated as 7.59 × 104 - 3.26 × 104 ) 4.33 × 104. h This is the CID conversion occurring before the FAIMS-MS orifice expressed as the percentage of the CID conversion occurring after FAIMS-MS orifice, calculated as (4.33 × 104/7.75 × 105) × 100 ) 5.6%. c

the m/z 617 ions upstream of the FAIMS system, is equal to 4.33 × 104 (7.59 × 104 - 3.26 × 104 ) 4.33 × 104). This corrected response amounts to 5.6% of the 7.75 × 105 ifetroban response seen in Figure 3a ((4.33 × 104/7.75 × 105) × 100 ) 5.6%), where the ifetroban response was attributed to the conversion of the Ife-Glu m/z 617 ions downstream 7842

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of the FAIMS system. Thus, the upstream conversion is insignificant compared to the downstream conversion occurring after the FAIMS-MS orifice region. It should be noted that the 2.94 min peak seen in the ifetroban SRM channel of Figure 4a is due to a minor presence of ifetroban as an impurity in the Ife-Glu sample used.

CONCLUSIONS We have demonstrated that FAIMS can be used to establish the location of the in-source CID in electrospray ionization mass spectrometry. For the ifetroban acylglucuronide that we investigated, the results show that the in-source CID, which is due to the loss of the glucuronide moiety, takes place almost entirely after the orifice of the mass spectrometer. Under the circumstance, it may be more appropriate to describe such a conversion of a precursor ion to its product ion as “post-orifice CID” rather than “in-source CID”. The technique described here can be used to determine the location of the in-source CID exhibited by a

variety of compounds in mass spectrometers equipped with not only electrospray ionization but also atmospheric pressurechemical-ionization sources. Establishing the location and the parameters that control in-source CID would help in controlling its occurrence. Minimizing the in-source CID of prodrugs or metabolites could be essential in developing bioanalytical methods used for quantitation of parent drugs in biological matrices.

Received for review June 5, 2009. Accepted July 29, 2009. AC9012336

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