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LC-FAIMS-MS/MS for Quantification of a Peptide in Plasma and Evaluation of FAIMS Global Selectivity from Plasma Components Yuan-Qing Xia,* Steven T. Wu, and Mohammed Jemal* Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey 08543 As a continuation of the evaluation of the utility of highfield asymmetric waveform ion mobility spectrometry (FAIMS) in quantitative bioanalysis, we have developed a sensitive and selective method for the quantification of a peptide drug candidate in rat plasma using FAIMS coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The LC-FAIMS-MS/MS method provided significant advantage over the corresponding LCMS/MS method by reducing chemical/endogenous background noise associated with plasma matrix, thereby improving the sensitivity via increasing the signal-to-noise ratio. Linearity was established within 1-1000 nM in rat plasma, and the overall method accuracy and precision were good meeting the generally adopted acceptance criteria for a bioanalytical method. In a related investigation, we demonstrated the global selectivity of FAIMS from plasma endogenous components as a function of the compensation voltage (CV) across molecular masses that encompass small-molecule drugs. This work demonstrates that FAIMS coupled with LC-MS/MS can be highly advantageous in quantitative bioanalysis. An emerging technique known as high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) has been demonstrated to reduce chemical noise, enhance analyte detection, eliminate interfering metabolites and prodrugs, and separate isomeric analytes.1–19 The FAIMS device, located be* To whom correspondence should be addressed. Yuan-Qing Xia: e-mail,
[email protected]; phone, 609-252-3067; fax, 609-252-3315. Mohammed Jemal: e-mail,
[email protected]; phone, 609-252-3572; fax, 609-252-3315. (1) Guevremont, R. J. Chromatogr., A 2004, 1058, 3. (2) Shvartsburg, A. A.; Tang, K.; Smith, R. D. Anal. Chem. 2004, 76, 7366. (3) Shvartsburg, A. A.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2005, 16, 2. (4) Shvartsburg, A. A.; Li, F.; Tang, K.; Smith, R. D. Anal. Chem. 2006, 78, 3706. (5) Kolakoswski, B. M.; McCooeye, M. A.; Mester, Z. Rapid Commun. Mass Spectrom. 2006, 20, 3319. (6) Barnett, D. A.; Ells, B.; Guevremont, R.; Purves, R. W. J. Am. Soc. Mass Spectrom. 1999, 10, 1279. (7) McCooeye, M.; Ding, L.; Gardner, G. J.; Fraser, C. A.; Lam, J.; Sturgeon, R. E.; Mester, Z. Anal. Chem. 2003, 75, 2538. (8) McCooeye, M. A.; Mester, Z.; Ells, B.; Barnett, D. A.; Purves, R. W.; Guevremont, R. Anal. Chem. 2002, 74, 3071. (9) Cui, M.; Ding, L.; Mester, Z. Anal. Chem. 2003, 75, 5847. (10) Kapron, T.; Jemal, M.; Duncan, G.; Kolakowski, B.; Purves, R. Rapid Commun. Mass Spectrom. 2005, 19, 1979. 10.1021/ac8010846 CCC: $40.75 2008 American Chemical Society Published on Web 07/25/2008
tween the ion source and the entrance of the mass spectrometer, is operated under atmospheric pressure. Detailed discussions of the fundamental principles of FAIMS have been presented in previous publications.1–5 In brief, the separation of ions is based on analyte-specific differences in ion mobility under the influence of a high electric field, known as the dispersion voltage (DV), and a low electric field of a waveform applied to the inner and outer electrodes. Ions drift from their straight paths as they travel between the electrodes. If uncorrected, the ions will have a drift in every waveform cycle and eventually hit the walls of the electrodes and discharge. A low direct-current voltage, known as the compensation voltage (CV), is applied to reverse the drift. The CV thus controls which ions are transmitted through FAIMS, and gas is used to propel the ions through the electrodes. The resolution of ions by FAIMS is based on the difference between the CV values of the ions. FAIMS, when used in conjunction with LC-MS/MS, acts as a postcolumn, pre-mass spectrometer ion filter in which only selected ions generated from electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) sources are transmitted. Recently, we reported the development of an LC-FAIMS-MS/ MS bioanalytical method for the simultaneous quantification of multiple small-molecule analytes in plasma, using nefazodone and its metabolites as model compounds.12 As a continuation of exploring FAIMS in quantitative bioanalysis, we evaluated its use in the quantitative determination of peptides in plasma. In this paper, we present the development of an LC-FAIMS-selected reaction monitoring (SRM) method for quantification of a peptide drug candidate in rat plasma. The LC-FAIMS-SRM method is compared with a conventional LC-SRM method for sensitivity, selectivity, and reproducibility. In addition, we present the outcomes of our investigation of the selectivity from plasma (11) Kapron, J.; Wu, J.; Mauriala, T.; Clark, P.; Purves, R. W.; Bateman, K. P. Rapid Commun. Mass Spectrom. 2006, 20, 1504. (12) Wu, S. T.; Xia, Y.-Q.; Jemal, M. Rapid Commun. Mass Spectrom. 2007, 21, 3667. (13) McCooeye, M.; Mester, Z. Rapid Commun. Mass Spectrom. 2006, 20, 1801. (14) Venne, K.; Bonneil, E.; Eng, K.; Thibault, P. Anal. Chem. 2005, 77, 2176. (15) Gabryelski, W.; Froese, K. L. J. Am. Soc. Mass Spectrom. 2003, 14, 265. (16) Borysik, A. J. H.; Read, P.; Little, A. R.; Bateman, R. H.; Radford, S. E.; Ashcroft, A. E. Rapid Commun. Mass Spectrom. 2004, 18, 2229. (17) Robinson, E. W.; Williams, E. R. J. Am. Soc. Mass Spectrom. 2005, 16, 1427. (18) Tang, K.; Li, F.; Shvartsburg, A. A.; Strittmatter, E. F.; Smith, R. D. Anal. Chem. 2005, 77, 6381. (19) Sultan, J.; Gabryelski, W. Anal. Chem. 2006, 78, 2905.
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Table 1. FAIMS Parameters Used parameters
I
II
compensation voltage (V) dispersion voltage (V) total gas flow (helium/ nitrogen, L/min) % helium/nitrogen inner/outer electrode temperatures (°C) outer bias voltage (V)
-21.9 -5000 3.2 40/60 50/90 35
-18.3 -5000 3.2 40/60 50/90 35
endogenous components that FAIMS provides for analytes of different molecular masses under different CV values. EXPERIMENTAL SECTION Instrument and Chemicals. A Thermo Finnigan TSQ Quantum Ultra mass spectrometer equipped with a heated-electrospray ionization (HESI) source and FAIMS device (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 mm × 50 mm, 5 µm), was purchased from Waters (Milford, MA). Peptides I and II (the internal standard) were proprietary products of Bristol-Myers Squibb Company (Princeton, NJ). I has a molecular weight of 1598.72 and elemental composition of C78H96FN15O21, and II has a molecular weight of 1605.74 and elemental composition of C80H97FN16O19. Ammonium bicarbonate (>99%) was purchased from Sigma-Aldrich (St. Louis, MO). HPLC-grade acetonitrile was obtained from EM Science (Gibbstown, NJ). House nitrogen (99.99%) was used; helium (ultra high purity) and argon (99.99%) were purchased from Airgas (Radnor, PA). Drug-free rat plasma, containing disodium-EDTA as the anticoagulant, was obtained from Bioreclamation, Inc. (Hicksville, NY). Mobile phase A was water/acetonitrile (90/10,
v/v), and mobile phase B was water/acetonitrile (10/90, v/v), each containing 5 mM ammonium bicarbonate. Preparation of Standard and QC Samples. Stock solutions of I and II were prepared in acetonitrile/water (90/10, v/v) at a concentration of 1.00 mM. Separate weighings of I were used for the preparation of the standards and quality control (QC) samples. Standard working solutions of I were prepared by the dilution of the stock solution with acetonitrile/water (50/50, v/v). Standard curve samples were prepared by adding 5 µL of a standard working solution of I into 50 µL of blank rat plasma to obtain concentrations of 1, 2, 10, 50, 250, 500, and 1000 nM of I. QC samples of I were prepared by adding the analyte into blank rat plasma to obtain concentrations of 1, 3, 500, and 800 nM. The internal standard solution, containing 200 nM of II, was prepared in acetonitrile/water (50/50, v/v). Sample Extraction. An aliquot (25 µL) of the internal standard working solution was added to 50 µL of each standard or QC sample, with each standard level in duplicate and each QC level in five replicates. An aliquot of 0.15 mL of acetonitrile was then added to each well and mixed for 5 min, followed by centrifugation for 10 min at 3000g at 10 °C. The supernatant from each sample was transferred to a clean 96-well plate and evaporated to dryness under a stream of nitrogen at room temperature. The residues were dissolved in 0.1 mL of reconstitution solution consisting of water and acetonitrile (50:50, v/v). The plate was sealed and placed on the autosampler for analysis. LC-FAIMS-MS/MS Conditions. Analyte CV was determined by infusing 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 operated in the positive ESI mode. The CV was scanned from 0 to -30 V in 3.9 min. The
Figure 1. Comparison of the full scan mass spectra (average) across the chromatographic retention window of 2.4-2.6 min obtained with (a) LC-MS and (b) LC-FAIMS-MS using the acetonitrile-precipitated supernatant of rat plasma containing I (0.5 µM). The FAIMS conditions were the same as in Table 1 with the CV set at -21.9 V. 7138
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Figure 2. Positive full scan FAIMS-MS mass spectra (average) obtained with the infusion of the acetonitrile-precipitated supernatant of blank rat plasma using different CV values: (a) only MS without FAIMS (presented as the baseline reference); (b) CV ) 0 V; (c) CV ) -5 V; (d) CV ) -10 V; (e) CV ) -15 V; (f) CV ) -20 V; (g) CV ) -25 V. The other FAIMS conditions used were as listed in Table 1.
ESI voltage was set at 4200 V. The heated electrospray vapor temperature and the heated capillary temperature were at 350 and 300 °C, respectively. The collision energy values used were 15 and 30 eV for I and II, respectively. Other parameters, such as capillary and tube lens offsets, were optimized for each SRM transition. Chromatographic data were acquired and processed using Thermo Finnigan Xcalibur 2.0 SR2, which was also used to control the FAIMS. LCquan 2.5 was used for integration and quantitation using a linear regression weighted to 1/x. The scan time was 0.05 s, and the scan width was 0.5 Da for each SRM transition. An Atlantis dC18 column (2.1 mm × 50 mm, 5 µm) was used for chromatographic separation with a gradient elution using mobile phases A and B at a flow rate of 0.4 mL/min. The gradient elution consisted of a linear increase from 20% B to 65% B in 3 min, with the subsequent ramping up to 95% B in 0.1 min and then holding there for 1 min. The gradient was then returned to initial conditions, for a total run time of 5 min. The column temperature was maintained at 30 °C, and the injection volume was 5 µL. Method Comparison. The same sets of standard curve and QC samples were analyzed using LC-FAIMS-SRM and LC-SRM methods. The standard curve linearity, accuracy, and precision for each method were evaluated using three separate runs, with each run consisting of a standard curve and QC samples. The standard curve ranged from 1 to 1000 nM, with each standard sample run in duplicate. Four levels of QC samples were used, with each level analyzed in quintuplicate. RESULTS AND DISCUSSION
Figure 3. Negative full scan FAIMS-MS mass spectra (average) obtained with the infusion of the acetonitrile-precipitated supernatant of blank rat plasma using different CV values: (a) only MS without FAIMS (presented as the baseline reference); (b) CV ) 0 V; (c) CV ) 10 V; (d) CV ) 15 V; (e) CV ) 20 V; (f) CV ) 25 V; (g) CV ) 30 V. The other FAIMS conditions used were as listed in Table 1.
final FAIMS parameters used are listed in Table 1. For selected reaction monitoring (SRM), the transitions m/z 800.3 f m/z 523.2 and m/z 803.8 f m/z 256.1 were used for I and II, respectively. For the ESI, nitrogen was used as both the sheath and auxiliary gas and was set to 60 and 55 psi, respectively. Argon was used as the collision gas and set to 1.0 mTorr. The
One of the features of FAIMS is its ability to reduce chemical/ endogenous ions, thereby increasing the sensitivity via improving the signal-to-noise (S/N) ratio. Accordingly, we compared the endogenous ions of a crude extract (acetonitrile-protein precipitation) of rat plasma containing I (0.5 µM) using LC-MS and LCFAIMS-MS (CV at -21.9) systems. The MS scan range was from m/z 100 to m/z 1500 (scanned in 2 s). The average full scan mass spectra from the two systems were compared in the retention time window between 2.4 and 2.6 min, where I elutes. More low-mass ions (
