Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6790–6796 Improved Sensitivity Mass Spectrometric Detection of Eicosanoids by Charge Reversal Derivatization James G. Bollinger, † Wallace Thompson, † Ying Lai, ‡ Rob C. Oslund, † Teal S. Hallstrand, ‡ Martin Sadilek, † Frantisek Turecek, † and Michael H. Gelb* ,†,§ Departments of Chemistry, Medicine, and Biochemistry, University of Washington, Seattle, Washington 98195 Combined liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is a powerful method for the analysis of oxygenated metabolites of polyunsaturated fatty acids including eicosanoids. Here we describe the synthesis of a new derivatization reagent N-(4-aminomethylphenyl)pyridinium (AMPP) that can be coupled to eicosanoids via an amide linkage in quantitative yield. Conversion of the carboxylic acid of eicosanoids to a cationic AMPP amide improves sensitivity of detection by 10- to 20-fold compared to negative mode electrospray ionization detection of underivatized analytes. This charge reversal derivatization allows detection of cations rather than anions in the electrospray ionization mass spectrometer, which enhances sensitivity. Another factor is that AMPP amides undergo considerable collision-induced dissociation in the analyte portion rather than exclusively in the cationic tag portion, which allows isobaric derivatives to be distinguished by tandem mass spectrometry, and this further enhances sensitivity and specificity. This simple derivatization method allows prostaglandins, thromboxane B2, leukotriene B4, hydroxyeicosatetraenoic acid isomers, and arachidonic acid to be quantified in complex biological samples with limits of quantification in the 200-900 fg range. One can anticipate that the AMPP derivatization method can be extended to other carboxylic acid analytes for enhanced sensitivity detection. Liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has emerged as a powerful method to detect oxygenated derivatives of fatty acids including eicosanoids (for example, refs 1-3). With these methods it is possible to analyze a large collection of eicosanoids in a single LC-ESI-MS/MS run. These lipid mediators are detected by single * To whom correspondence should be addressed. Michael H. Gelb, Depts. of Chemistry and Biochemistry, Campus Box 35100, University of Washington, Seattle, WA 98195. Phone: 206 525-8405. Fax: 206 685-8665. E-mail: gelb@chem.washington.edu. † Department of Chemistry. ‡ Department of Medicine. § Department of Biochemistry. (1) Dickinson, J. S.; Murphy, R. C. J. Am. Soc. Mass Spectrom. 2002, 13, 1227. (2) Kita, Y.; Takahashi, T.; Uozumi, N.; Nallan, L.; Gelb, M. H.; Shimizu, T. Biochem. Biophys. Res. Commun. 2005, 330, 898. (3) Buczynski, M. W.; Stephens, D. L.; Bowers-Gentry, R. C.; Grkovich, A.; Deems, R. A.; Dennis, E. A. J. Biol. Chem. 2007, 282, 22834. 6790 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 reaction monitoring (SRM) in which precursor ions are isolated in the first stage of the mass spectrometer followed by collisioninduced dissociation to give fragment ions, which are detected after an additional stage of mass spectrometer isolation. The current limit of quantification for these analytes is in the ∼10-20 pg range. This sensitivity level is appropriate for studies with cultured cells in vitro or with relatively large tissue samples, but it is not sufficient for studies with smaller volume samples such as joint synovial fluid or bronchoalveolar lavage fluid from experimental rodents. Given the importance of oxygenated fatty acid derivatives in numerous medically important processes such as inflammation and resolution of inflammation, we sought to improve the LC-ESI-MS/MS sensitivity of detection of these lipid mediators using a widely available analytical platform. For reasons that are not well understood, cations generally form gaseous ions better than anions in the electrospray ionization source of the mass spectrometer. Additionally, for underivatized carboxylic acids it is required to add a weak organic acid to the chromatographic mobile phase, i.e., formic acid, so that the carboxylic acid is kept in its protonated state, which allows it to be retained on the reverse-phase column to ensure chromatographic separation. However, the presence of the weak acid offsets the formation of carboxylate anions in the electrospray source because the weak acid carries most of the anionic charge in the electrospray droplets, and thus formation of analyte anions is suppressed. We reasoned that conversion of the carboxylic acid to a fixed-charge cationic derivative would lead to improved detection sensitivity by ESI-MS/MS. Charge-reversal derivatization of carboxylic acids with quaternary amines has been explored in previous work (for example, refs 4-6). However, these reagents utilize organic cations that tend to fragment by collision-induced dissociation near the cationic site. Fragmentation in the derivatization tag is not desirable because analytes that form isobaric precursor ions and that comigrate on the LC column will not be distinguished in the mass spectrometer if they give rise to the same detected fragment ion. This loss of analytical specificity is a serious problem when analyzing complex biological samples. Fragmentation in the analyte portion rather than in the tag portion (4) Lamos, S. M.; Shortreed, M. R.; Frey, B. L.; Belshaw, P. J.; Smith, L. M. Anal. Chem. 2007, 79, 5143. (5) Yang, W.; Adamec, J.; Regnier, F. E. Anal. Chem. 2007, 79, 5150. (6) Pettinella, C.; Lee, S. H.; Cipollone, F.; Blair, I. A. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2007, 850, 168. 10.1021/ac100720p © 2010 American Chemical Society Published on Web 07/22/2010
also reduces chemical noise, which also enhances sensitivity of detection. In this study we report the design and synthesis of a new cationic tag and show that it can be quantitatively attached via an amide linkage to the carboxyl group of eicosanoids by a simple derivatization procedure. We then show that the derivatized eicosanoids can be analyzed by LC-ESI-MS/MS with limits of quantification that are well below those reported for underivatized eicosanoids. EXPERIMENTAL METHODS Synthesis of AMPP. Pyridine (40 mmol, 3.2 mL) was dissolved in 46 mL of absolute ethanol followed by the addition of 1-chloro-2,4-dinitrobenzene (40 mmol, 8.2 g, Aldrich). The mixture was heated with a reflux condenser at 98 °C for 16 h under nitrogen. After cooling, ethanol was removed by rotary evaporation, and the crude product was recrystallized by dissolving in a minimal amount of hot ethanol and allowing the solution to slowly cool. The product N-2,4 dinitrophenyl pyridinium chloride was isolated as a yellow solid in 62% yield, and its identity was confirmed by melting point analysis (189-191 °C observed, 189-190 °C reported 7 ). N-2,4-Dinitrophenyl pyridinium chloride (16.8 mmol, 4.76 g) was dissolved in 70 mL of ethanol-pyridine (3:1). 4-[(N-Boc) -amino-methyl] aniline (33.6 mmol, 7.56 g, Aldrich) was added, and the reaction mixture was heated under a reflux condenser at 98 °C under nitrogen for 3 h. After cooling, 700 mL of water was added to precipitate 2,4-dinitroaniline. After filtration, the filtrate was concentrated to dryness by rotary evaporation, and the product was isolated as a brown oil. This oil was treated with 112 mL of 25% (v/v) trifluoroacetic acid in dichloromethane for 30 min at room temperature. The mixture was concentrated by rotary evaporation, and the solid was triturated twice with benzene to remove excess trifluoroacetic acid. The mixture was again concentrated by rotary evaporation. The residue was dissolved in a minimal amount of heated ethanol, the solution was allowed to cool for ∼5 min, and then diethyl ether was added with swirling until the solution started to cloud up. The mixture was transferred to the freezer (-20 °C) and left overnight. The mixture was allowed to warm to room temperature and then decanted. The obtained mother liquor was treated with additional diethyl ether as above to give additional AMPP solid. The solids were combined and triturated with diethyl ether. The solid was dried under vacuum to give 3.30 g of AMPP as a brown solid, 59% yield. 1 H NMR (300 MHz, D6-DMSO) 9.34 (d, 2H), 8.81 (t, 1H), 8.53 (broad, 3H), 8.33 (t, 2H), 7.95 (d, 2H), 7.80 (d,2H), 4.22 (s, 2H) ( 1 H NMR spectra are shown in the Supporting Information, estimated purity of AMPP is >95%). Preparation of Eicosanoid Stock Solutions. The following eicosanoid standards from Cayman Chemicals were used (PGE2, PGD2, PGF2R, 6-keto-PGF1R, TxB2, 5(S)-HETE, 8(S)-HETE, 11(S)-HETE, 12(S)-HETE, 15(S)-HETE, LTB4, arachidonic acid, D4-PGE2, D4-PGD2, D4-PGF2R, D4-6-keto-PGF1R, D4-TXB2, D8- 5(S)-HETE, D4-LTB4, D8-arachidonic acid). Stock solutions of eicosanoids were prepared at a concentration of 100 pg/µL in absolute ethanol and stored at -80 °C under Ar in Teflon septum, screw cap vials. Serial dilutions of the stock solutions were made in absolute ethanol for standard curve and extrac- (7) Park, K. K.; Lee, J.; Han, D. Bull. Korean Chem. Soc. 1985, 6, 141. tion analysis. Internal standards were diluted to a working stock of 5 pg/µL in absolute ethanol. Preparation of Samples Prior to Derivatization with AMPP. Standard Curves. Each sample contained 50 pg of each internal standard and various amounts of nonisotopic eicosanoids (added from the stock solutions described above) transferred to a glass autosampler vial insert (Agilent catalog no. 5183-2085). Solvent was removed with a stream of nitrogen, and the residue was derivatized with AMPP as described below. Mouse Serum. Analysis of endogenous eicosanoids in serum was carried out with commercial mouse serum (Atlantic Biologicals catalog no. S18110). A volume of 1, 5, or 10 µL of serum was placed in a glass autosampler vial insert. Two volumes of methanol (LC/MS, JT Baker catalog no. 9863-01) containing 50 pg of each internal standard were added. The vial insert was mixed on a vortex mixer for ∼10 s. The concentration of methanol was lowered to 10% (v/v) by addition of purified water (Milli-Q, Millipore Corp.), and the samples were loaded via a glass Pasteur pipet onto a solid phase extraction cartridge (10 mg Oasis-HLB, Waters catalog no. 186000383). The cartridges were previously washed with 1 mL of methanol and then 2 × 0.75 mL of 95:5 water-methanol. After sample loading, the sample tube was rinsed with 200 µL of purified water-methanol (95:5, v/v), and this was added to the cartridge. The cartridge was washed with 2 × 1mL of water-methanol (95:5, v/v). Additional solvent was forced out of the cartridge solid phase by applying medium pressure N2 (house N2 passed through a 0.2 µm cartridge filter) for a few seconds. Column eluant receiver vials (Waters Total Recovery autosampler vials, Waters catalog no. 186002805) were placed under the cartridges. The cartridges were then eluted with methanol (1 mL). All cartridge steps were carried out using a vacuum manifold (Waters catalog no. WAT200606) attached to a water aspirator. Solvent was removed by placing the receiver vials in a centrifugal evaporator (Speed-Vac). These processed samples were derivatized with AMPP (see below) without storage. Lung Epithelial Cells. The University of Washington Institutional Review Board approved the studies involving human subjects, and written informed consent was obtained from all participants. Primary bronchial epithelial cells were isolated from a volunteer with asthma during a bronchoscopy using a nylon cytology brush of cells from subsegmental airways. To establish primary culture, the epithelial cells were seeded into a culture vessel coated with type 1 collagen in bronchial epithelial basal media (BEBM, Lonza, Allendale, NJ) supplemented with bovine pituitary extract, insulin, hydrocortisone, gentamicin, amphotericin B, fluconazole, retinoic acid, transferrin, triiodothyronine, epinephrine, and human recombinant epidermal growth factor (serum-free BEGM) and maintained at 37 °C in a humidified incubator. After expansion in vitro, passage 2 epithelial cells were grown to >90% confluence on a 12-well plate. The medium was changed to 200 µL of Hanks balanced salt solution, and the cells were treated with either calcium ionophore (A23187, 10 µM in DMSO) or a DMSO-containing control solution for 20 min at 37 °C. The synthesis of eicosanoids was stopped by the addition of 4 volumes of ice-cold methanol with 0.2% formic acid, and samples were stored at -80 °C until processed. The number of epithelial Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6791
Page 1 and 2: Anal. Chem. 2010, 82, 6745-6750 Let
Page 3 and 4: purchased from Invitrogen-Molecular
Page 5 and 6: Figure 3. Electropherograms of TPP-
Page 7 and 8: Anal. Chem. 2010, 82, 6751-6755 Res
Page 9 and 10: (pH 8.0) with cysteine and cystamin
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
Page 15 and 16: coater at 2000 rpm for 20 s, and th
Page 17 and 18: Figure 5. Comparison of cyclic volt
Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
Page 23 and 24: Figure 1. GC/MS total ion chromatog
Page 25 and 26: Table 2. Concentrations and Stable
Page 27 and 28: on a substrate are preferred. 20-24
Page 29 and 30: Figure 4. SERS analysis of NAADP co
Page 31 and 32: Anal. Chem. 2010, 82, 6775-6781 Hig
Page 33 and 34: tion, 2 µL of proprionaldehyde wer
Page 35 and 36: Figure 4. Analysis of 2a by HPLC-MS
Page 37 and 38: eaction of 1a with PBH can be condu
Page 39 and 40: Numerous references had demonstrate
Page 41 and 42: Figure 1. TEM images of the prepare
Page 43 and 44: Figure 4. Schematic representation
Page 45: esult in a big SPR signal change wi
Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
Page 53 and 54: Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56: Figure 1. Schematic view of the pre
Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
Page 61 and 62: Table 3. Ambient Measurement Result
Page 63 and 64: Anal. Chem. 2010, 82, 6807-6813 Dir
Page 65 and 66: Polymerase (1 U per sample). Reacti
Page 67 and 68: of which was constant for all ampli
Page 69 and 70: analytically useful signals at less
Page 71 and 72: carbon black and RP-C18 for the ext
Page 73 and 74: solution in an equal volume, and 1
Page 75 and 76: Table 2. Concentrations and Ratios
Page 77 and 78: Anal. Chem. 2010, 82, 6821-6829 Mac
Page 79 and 80: mg/mL protein, followed by separati
Page 81 and 82: Figure 2. Productivity of SEQUST an
Page 83 and 84: Figure 4. High-resolution MS/MS spe
Page 85 and 86: Figure 6. Characterization of PSMs
Page 87 and 88: containing T-T mismatches. 23 Based
Page 89 and 90: Figure 1. Extinction spectra of sol
Page 91 and 92: Figure 3. (A) The value of Ex650 nm
Page 93 and 94: Figure 5. Extinction spectra and co
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constant medium for separation; we
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Figure 2. Standards of (Pi)n, n ) 1
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Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
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Figure 1. 226 Ra spectrum by liquid
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Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
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Figure 5. Volcano plots detailing t
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data as well. The 41 genes in Table
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corresponding compound if its chemi
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Figure 1. Schematic illustration of
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Table 1. Absolute Quantification Re
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ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
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Figure 5. Device to eject and mix s
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Anal. Chem. 2010, 82, 6877-6886 Imm
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NaCl, phosphate buffer saline (PBS)
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Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
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Table 1. Intraday Precision and Acc
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Anal. Chem. 2010, 82, 6887-6894 Fer
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
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Anal. Chem. 2010, 82, 6895-6903 Ele
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Table 1. Chemical Structure, pKa Va
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pH with a tilted baseline (Figure 1
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Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
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the multielement capabilities, the
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RESULTS AND DISCUSSION Sulfur Detec
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Table 2. Molecular Properties and C
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Anal. Chem. 2010, 82, 6911-6918 Dir
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
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Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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Figure 5. Extracted ion current ESI
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m/z 1172.935 was observed for Ser14
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linked products via affinity tags.
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Scheme 2. Fragmentation Mechanism o
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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Figure 5. (A) MALDI-TOF/TOF product
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
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allow the use of higher aptamer con
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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
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Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
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Table 2. Capillary Electrophoresis
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Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
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Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
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Anal. Chem. 2010, 82, 7015-7020 Sel
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Table 1. Effect of 1 D-LC Condition
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Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
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luciferase (T7 control vector) was
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Figure 3. Inhibitory effects of luc
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Anal. Chem. 2010, 82, 7027-7034 Pat
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Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
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Table 3. Film Thickness Measurement
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Anal. Chem. 2010, 82, 7035-7043 Qua
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Figure 1. (A) FL spectra of BSPOTPE
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Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
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Scheme 1. Proposed Mechanism for Fl
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one or more drawbacks including poo
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Figure 2. Free fluorophores do not
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Anal. Chem. 2010, 82, 7049-7052 Dev
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Figure 2. Comparative analysis of d
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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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