Anal. Chem. 1999, 71, 5481-5492
Effects of Mobile-Phase Additives, Solution pH, Ionization Constant, and Analyte Concentration on the Sensitivities and Electrospray Ionization Mass Spectra of Nucleoside Antiviral Agents Amin M. Kamel*
Department of Drug Metabolism, Central Research Division, Pfizer Inc., Groton, Connecticut 06340 Phyllis R. Brown
Department of Chemistry, University of Rhode Island, Kingston, Rhode Island 02881 Burnaby Munson
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
The effects of various mobile-phase additives, solution pH, pKa, and analyte concentration on electrospray ionization mass spectra of a series of purine and pyrimidine nucleoside antiviral agents were studied in both positive and negative ion modes. The use of 1% acetic acid resulted in good HPLC separation and the greatest sensitivity for [M + H]+ ions. In the negative ion mode, 50 mM ammonium hydroxide gave the greatest sensitivity for [M - H]- ions. The sensitivities as [M + H]+ ions were significantly larger than the sensitivities as [M - H]- ions for purine antiviral agents. Vidarabine monophosphate and pyrimidine antiviral agents, however, showed comparable or greater sensitivities as [M - H]- ions. The sensitivity as [M + H]+ showed no systematic variation with pH; however, the sensitivity as [M - H]- did increase with increasing pH. At constant pH, the ion intensity of the protonated species increased with increasing pKa. At higher analyte concentrations, dimer (M2H+) and trimer (M3H+) ions were observed. [M + Na]+ adducts were the dominant ions with 0.5 mM sodium salts for these compounds. The spectra of the more basic purine antiviral agents showed no [M + NH4]+ adduct ions, but [M + NH4]+ ions were the major peaks in the spectra of the less basic pyrimidine antiviral agents with ammonium salts. The ammonium adduct ion was formed preferentially when the proton affinity of the analyte was close to that of NH3. Abundant [M + OAc]ions were observed for all of the antiviral agents except vidarabine monophosphate from solutions with added HOAc, NaOAc, and NH4OAc. The utility of mobile phases containing 1% HOAc or 50 mM NH4OH was demonstrated for chromatographic separations. Atmospheric pressure ionization (API) methods, in particular electrospray ionization (ESI), used with liquid chromatography/ * Corresponding author: (tel) 860-441-1848; (fax) 860-715-7588; (e-mail)
[email protected]. 10.1021/ac9906429 CCC: $18.00 Published on Web 11/04/1999
© 1999 American Chemical Society
mass spectrometry (LC/MS) have revolutionized trace analyses for quantitative and qualitative studies in chemical and biological sciences.1,2 The unique ability of the ESI process to transfer compounds such as nucleic acids, peptides, proteins, carbohydrates, and other thermally labile compounds from the liquid phase to the gas phase as ions distinguishes this technique from other mass spectrometric ionization methods. In addition to increasing the applicability of LC/ESI-MS, major interests lie in achieving a better understanding of the ESI processes: the mechanisms of ionization, as well as the effects of experimental parameters on the ions that are produced. Parameters that affect the generation of ESI ions include mobilephase additives, solution pH, flow rate, solvent composition, and analyte concentration.3-9 The effects of several common solvents on ionization efficiency (or sensitivity), analyte charge state, and stability in negative ion ESI-MS have been examined.3,10 Electrospray response in various mobile-phase combinations has been also reported showing that the higher the percentage of organic component in the solvent system, the higher the ESI response.11 Several solvent properties, such as surface tension, conductivity, viscosity, and dielectric constant, were determined to be important parameters in the success of the ESI-MS process.12,13 The positive and negative ion electrospray responses in mobile phases that (1) Covey, T. R.; Lee, E. D.; Bruins, A. P.; Henion, J. D. Anal. Chem. 1986, 58 (14), 1451A-61A. (2) Bruins, A. P. Mass Spectrom. Rev. 1991, 10 (1), 53-77. (3) Straub, R. F.; Voyksner, R. D. J. Am. Soc. Mass Spectrom. 1993, 4 (7), 57887. (4) Apffel, A.; Fischer, S.; Goldberg, G.; Goodley, P. C.; Kuhlmann, F. E. J. Chromatogr., A 1995, 712 (1), 177-90. (5) Tang, L.; Kebarle, P. Anal. Chem. 1993, 65 (24), 3654-68. (6) Tang, L.; Kebarle, P. Anal. Chem. 1991, 63 (23), 2709-15. (7) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1991, 63 (18), 1989-98. (8) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1990, 62 (9), 957-67. (9) Sunner, J.; Nicol, G.; Kebarle, P. Anal. Chem. 1988, 60 (13), 1300-7. (10) Cole, R. B.; Harrata, A. K. J. Am. Soc. Mass Spectrom. 1993, 4 (7), 546-56. (11) Straub, R. F.; Voyksner, R. D. J. Chromatogr. 1993, 647 (1), 167-81. (12) Hayati, I; Bailey, A. I.; Tadros, T. F. Nature 1986, 319, 41-3.
Analytical Chemistry, Vol. 71, No. 24, December 15, 1999 5481
contained methanol or acetonitrile at neutral and low pH and with and without ammonium acetate were investigated.14 The positive ion response as [M + H]+ or [M + NH4]+ was consistently higher in a methanolic mobile phase under all conditions studied. The formation of dimer and trimer ions of nucleosides has been reported at concentrations of ∼100 µM, and the ion signals of protonated species were reported to increase with increasing pKa.15 Gas-phase basicity of the analytes, pKa, and the nature of the electrolyte in solution have been reported to play an important role in the electrospray ionization of selected nucleobases and nucleosides. The intensity of MH+ ions for compounds with pKa e 4 was proportional to their pKa. For compounds with pKa < 3, the addition of ammonium salts was reported to promote gasphase proton-transfer reactions and to increase the MH+ ion intensity.16 Electrospray ion currents of salts increase linearly with increasing concentrations to an approximately constant value, independent of concentration or nature of salt.5 A number of antiviral agents have been developed and used against a wide variety of DNA and RNA viruses. Zidovudine (3′azido-3′-deoxythymidine, AZT) is probably the most widely used antiviral agent in the acquired immunodeficiency syndrome (AIDS) treatment.17,18 Due to dose-dependent toxic effects,19 highly sensitive analyses are required to determine the plasma concentration of AZT. Antiviral agents, which are analogues of naturally occurring nucleosides and nucleotides, have been studied extensively by mass spectrometry.20-23 Quantitative and qualitative determinations of drugs and their metabolites in biological fluids are essential in the pharmaceutical and biotechnology industries. Highly sensitive and accurate assays are required for routine analyses of clinical samples, pharmacokinetic and toxicokinetic studies, and structure elucidation of drug metabolites. The objective of this study was to investigate the effects of various mobile-phase additives, solution pH, ionization constant, and analyte concentration on the ESI mass spectra and molar responses (or sensitivities) of a series of structurally related compounds. Mobile-phase additives are used to improve chromatographic separations of complex mixtures, to increase the solubilities of the analytes, to improve the ESI responses of the analytes, or to improve ESI performance. Except for NaOAc and NaI, the mobile-phase additives under investigation are suitable (13) Tyczkowska, K. L.; Voyksner, R. D.; Aronson, A. L. J. Chromatogr. 1992, 594, 195-201. (14) Jemal, M.; Hawthorne, D. J. Rapid Commun. Mass Spectrom. 1999, 13 (1), 61-6. (15) Banks, J. F., Jr.; Shen, S.; Whitehouse, C. M.; Fenn, J. B. Anal. Chem. 1994, 66 (3), 406-14. (16) Yen, T.-Y.; Charles, M. J.; Voyksner, R. D. J. Am. Soc. Mass Spectrom. 1996, 7 (11), 1106-1108. (17) Nakashima, H.; Matsui, T.; Harada, S.; Kobayashi, N.; Matsuda, A.; Ueda, T.; Yamamoto, N. Antimicrob. Agents Chemother. 1986, 30 (6), 933-7. (18) Chaisson, R. E.; Allain, J. P; Leuther, M.; Volberding, P. A. N. Engl. J. Med. 1986, 315 (25), 1610-1. (19) Richman, D. D.; Fischl, M. A.; Grieco, M. H.; Gottlieb, M. S.; Volberding, P. A.; Laskin, O. L.; Leedom, J. M.; Groopman, J. E.; Mildvan, D.; Hirsch, M. S. N. Engl. J. Med. 1987, 317 (4), 192-7. (20) Buchanan, M. V.; Hettich, R. L.; Stemmler, E. Book of Abstracts, 213th ACS National Meeting, San Francisco, April 13-17, 1997. (21) Zhao, Z; Fuciarelli, A. F.; Smith, R. D. J. Capillary Electrophor. 1996, 3 (2), 111-6. (22) Porsburgh, M. J.; Foster, T. J.; Barth, P. T.; Coggins, J. R. Microbiology 1996, 142 (10), 2943-50. (23) Ye, M. Y.; Shen, Y. J. Liq. Chromatogr. 1994, 17 (4), 773-91.
5482
Analytical Chemistry, Vol. 71, No. 24, December 15, 1999
for reversed-phase chromatography and are volatile for mass spectrometric analysis. The less volatile salts, NaOAc and NaI, were used as mobile-phase additives to form sodium adduct ions in addition to the normally observed protonated species. Sodium salts are frequently present in samples and are often difficult to remove. The sodium adducts can be useful in the determination of molecular weights of unknown analytes24 and in quantitative analysis.25 Optimal conditions for chromatographic separations may not be the same as those for ESI-MS analysis. Chromatographic analyses of the purine and pyrimidine nucleoside antiviral agents under investigation were obtained using conditions that gave the best ESI-MS spectra. EXPERIMENTAL SECTION Materials. Seven antiviral agents (acycloguanosine, vidarabine, vidarabine monophosphate, trifluridine, 5-iodo-2′-deoxyuridine, 5-iodo-2′,3′ dideoxyuridine, AZT), methyl stearate, methyl oleate, pyrazole, and 1-aminodecane were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol (MeOH) and deionized water (HPLC grade) were obtained from J. T. Baker (Phillipsburg, NJ). The other compounds, all HPLC grade, were obtained from Fisher (Fair Lawn, NJ): trifluoroacetic acid (TFAH), acetic acid (HOAc), ammonium acetate (NH4OAc), sodium acetate (NaOAc), ammonium hydroxide (NH4OH), and sodium iodide (NaI). Mass Spectrometry and Sample Introduction. Mass spectral analyses were performed with a Sciex API-IIIplus triplequadrupole mass spectrometer with a mass range to 2400 µ (Thornhill, ON, Canada). The mass spectrometer was equipped with an ion spray (IS) interface set at a nebulizer gas pressure of nitrogen of 60 psi. The nitrogen curtain gas was adjusted to a constant flow rate of 1.2 L/min. Positive or negative ions formed at atmospheric pressure were sampled into the quadrupole mass filter via a 0.0045-in.-diameter aperture. To evaluate the effects of mobile-phase additives and analyte concentration on ionization efficiency, samples were infused into the electrospray interface using a Harvard syringe pump (South Natick, MA) at a flow rate of 5 µL/min. Electrospray ionization mass spectra were acquired in both positive and negative ion modes by scanning over the m/z range 150-700 µ in steps of 0.1 µ with a 2-ms dwell time. The signal was averaged over five scans. In the concentration-dependence studies, the mass range was extended to 1500 µ to investigate the formation of dimer and trimer ions. Voltages were the same in positive and negative ion modes except for the changes in the polarities. For chromatographic separations, samples were injected into the HPLC column. The column effluent was split and a flow of ∼30 µL/min was introduced into the ion spray ionization source. The quadrupole power supply was set for unit mass resolution. Collisionally induced dissociation (CID) studies were performed using argon at a thickness of ∼2 × 1015 atoms/cm2 and a collision energy of 25 eV. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode with a dwell time of 250 ms to monitor selected product ions of specific parent ions in the chromatographic analyses. The [M + H]+ or [M - H]- ions were collisionally dissociated in the second quadrupole. The nitrogen (24) Neubauer, G.; Anderegg, R. J. Anal. Chem. 1994, 66 (7), 1056-61. (25) Jemal, M.; Almond, R. B.; Teitz, D. S. Rapid Commun. Mass Spectrom. 1997, 11 (10), 1083-8.
flow rate, position of the ESI needle, infusion flow rate, and voltage settings were held constant during analyses. High-Pressure Liquid Chromatography. The high-performance liquid chromatography (HPLC) was carried out on a system composed of a Rheodyne injector (Cotati, CA) for manual injections and a HP-1050 solvent delivery system (Hewlett-Packard Co., Wilmington, DE). Chromatography was carried out on a Luna HPLC column (4.6 mm × 150 mm, 5-µm ODS, Phenomenex, Torrance, CA) with a mixture of methanol and 1% HOAc (pH ∼2.8) or 50 mM NH4OH (pH ∼10.1). The flow rate was established at 1 mL/min. The mobile phase consisted initially of 100% aqueous acetic acid (1%) or 50 mM NH4OH for 1 min and was then linearly changed to 100% methanol in 4 min. The chromatography was carried out under isocratic conditions for 3 min, and then the solvent composition was programmed back to the starting mobile phase in 2 min. The system was allowed to equilibrate for ∼5 min before making the next injection. Sample Preparation Procedure. The mobile-phase additive was added to water to give a solution of the desired concentration and the pH was measured. Equal volumes of each mobile-phase solution and methanol were mixed and the pH of the final solution was obtained. The latter pH value is reported in the tables. The two sets of pH measurements did not differ by more than (0.2 pH unit. Stock solutions (1.00 mg/mL) of the antiviral agents were prepared in methanol and stored under refrigeration. For the majority of experiments on sensitivity or ionization efficiency, a 50-µL aliquot of the sample in methanol was mixed with 2.45 mL of methanol. Then a volume of 2.50 mL of the mobilephase additive was added to give a final solution of 5.00 mL with a concentration of 10.0 µg/mL. The following mobile-phase additives were used: none (water, pH 6.4), 0.1% TFAH (pH 2.3), 1% HOAc (pH 3.1), 50 mM NH4OAc (pH 7.1), 0.5 mM NaOAc (pH 6.0), 5 mM NaOAc (pH 6.9), 50 mM NH4OH (pH 10.1), and 0.5 mM NaI (pH 6.9). Mixtures of 0.2 µg/mL of each analyte were prepared in 1% HOAc or 50 mM NH4OH/MeOH (9:1) and a 50µL aliquot was injected onto the column for the HPLC experiments. For the proton affinity study, methyl stearate, methyl oleate, pyrazole, and 1-aminodecane were dissolved or diluted with MeOH to give stock solutions with a concentration of 1 mg/mL. An aliquot (50 µL) of the sample in methanol was mixed with 2.45 mL of methanol. Then a volume of 2.50 mL of 50 mM NH4OH (pH 10.1) was added to give a final solution of 5.00 mL with a concentration of 10.0 µg/mL. RESULTS AND DISCUSSION The structures of seven purine and pyrimidine derivatives examined in this study are given in Table 1, together with their molecular weights and ionization constants, as pKa. There are two purine nucleosides, one purine nucleotide, and four pyrimidine nucleoside derivatives. Some of the pKa values are from the literature,26-32 and others were calculated with the ZPARC pKa program.33 There is a wide range in basicities of these compounds. (26) Izatt, R. M.; Christensen, J. J.; Rytting, J. H. Chem. Rev. 1971, 71 (5), 43982. (27) Lonnberg, H. In Biocoordination Chemistry; Burger, K., Ed.; Horwood: London, U.K., 1990; 284-346. (28) Shionoya, M.; Kimura, E.; Shiro, M. J. Am. Chem. Soc. 1993, 115 (15), 6730-7.
The purine derivatives are much more basic than the pyrimidine derivatives, as indicated by the pKa values for the dissociation of the monoprotonated species. The gas-phase proton affinities (PA) of these compounds are not known, but the proton affinities of guanosine and adenosine are ∼40 kJ/mol larger than the proton affinities of uridine and thymidine.34 Positive Ion ESI-MS. The positive ion ESI mass spectra for one of the purine derivatives, vidarabine, with the different mobilephase additives are shown in Figure 1. No significant fragment ions are observed. The dominant sample ions are [M + H]+ and [M + Na]+, with smaller amounts of dimer ions, [2M + H]+ and [2M + Na]+, at these relatively high concentrations of 10 µg/mL (37 µM). Sodiated ions, [M + Na]+, were observed in some solutions as seen in the MeOH/H2O spectrum of vidarabine in Figure 1. These spectra indicate the sensitivity of these ESI-MS spectra to impurities of cations. No significant abundances of multiply protonated ions were observed. Cluster ions, Na[NaOAc]n+, are the dominant species with 5.0 mM NaOAc, as noted previously with tetracyclines.35 The effects of mobile-phase additives on the intensities of [M + H]+ ions for the nucleoside antiviral agents are summarized in Table 2. The values for ion intensities were obtained from 10 µg/ mL solutions of each compound (28-44 µM). The intensities are the averages of five spectra obtained in rapid succession with a short-term precision of a few percent. Since the molecular weights of these compounds vary significantly and the ion intensity is a linear function of concentration in this concentration range, the values in Table 2 are reported as I/C, the ion intensity divided by the molar concentration, for comparison. The sensitivities as [M + H]+ for two purine derivatives, acycloguanosine and vidarabine, are similar but the sensitivity for vidarabine monophosphate in this MeOH/H2O mixture at pH ∼6.4 is much lower (∼4% of the sensitivity of vidarabine). The pKa1 values for acycloguanosine and vidarabine (∼2 and ∼4) correspond to the deprotonation of [M + H]+ ions to form the neutral molecules.26-27,30 Consequently, at pH ∼6, the dominant form of these species in solution is the neutral molecule. For vidarabine monophosphate, pKa1 ) 1.6 (calculated33) and pKa1 ) 1-2 for adenine monophosphate (AMP).26,27 These values correspond to the deprotonation of [M + H]+ from PO-H to form the neutral zwitterion; pKa2 (3.7 26,27 and 4.2 calculated33) corresponds to the deprotonation from N1H+ of the neutral zwitterion to form the monoanion; pKa3 (6.1-6.5 26-27,36) corresponds to deprotonation from the second hydrogen of the phosphate group of the monoanion to form the dianion. The dominant form of vidarabine monophosphate in solutions of pH 4-6 is the monoanion, and (29) Christensen, J. J.; Rytting, J. H.; Izatt, R. M. J. Phys. Chem. 1967, 71 (8), 2700-5. (30) Christensen, J. J.; Rytting, J. H.; Izatt, R. M. Biochemistry 1970, 9 (25), 4907-13. (31) Wataya, Y.; Sonobe, Y.; Maeda, M.; Yamaizumi, Z.; Aida, M.; Santi, D. V. J. Chem. Soc., Perkin Trans. 1 1987, (10), 2141-7. (32) Shionoya, M.; Ikeda, T.; Kimura, E.; Shiro, M. J. Am. Chem. Soc. 1994, 116 (9), 3848-59. (33) Molecular Modeling and Computational Chemistry Department, Pfizer Inc. ZPARC (Performs Automatic Reasoning in Chemistry) Version 1.1, ChemLogic Inc., July 1998. (34) The National Institute of Standards and Technology (NIST) Chemistry WebBook; NIST Standard Reference Database Number 69, August 1997. (35) Kamel, A.; Brown, P.; Munson, B. Anal. Chem. 1999, 71, 968-77. (36) Massoud, S. S.; Sigel, H. Inorg. Chem. 1988, 27 (8), 1447-53.
Analytical Chemistry, Vol. 71, No. 24, December 15, 1999
5483
Table 1. Structures and Ionization Constants of Antiviral Agents
a
Value calculated by ZPARC pKa program.33
b
Literature value (refs 26-32).
above pH ∼6, the dianion predominates; consequently, the greatly reduced sensitivity as [M + H]+ of vidarabine monophosphate compared with vidarabine is not surprising. 5484 Analytical Chemistry, Vol. 71, No. 24, December 15, 1999
In the MeOH/H2O solutions, the sensitivities of the pyrimidine derivatives as [M + H]+ ions are much lower than the sensitivities of the purine derivatives, ∼5-20%. These much lower sensitivities
Figure 1. Positive ion ESI spectra of vidarabine (MW ) 267) with different mobile-phase additives. Asterisk denotes the highest signal intensity for [M + H]+. Samples (10 ng/µL) were infused at 5 µL/min, and the signal was averaged over five scans.
for pyrimidine derivatives are consistent with their much weaker basicities in aqueous solution, as indicated by the pKa values for the deprotonation of the [M + H]+ ions that are given in Table 1:26-29,31-33 ∼- 5 for the pyrimidine derivatives and ∼2-4 for the purine derivatives. The sensitivities of all of these antiviral agents are larger with 0.1% TFAH as the mobile phase, pH 2.3, than with MeOH/H2O,
pH 6.4. The increases for acycloguanosine and vidarabine are relatively small (a factor of ∼1.3). At pH ∼2, the dominant solution form for these species should still be the neutral compound. There is, however, an increase of almost a factor of 10 in the sensitivity of vidarabine monophosphate. Qualitatively, one can attribute the large increase in sensitivity to the increase in concentration of neutral phosphate and protonated species because pKa2 {M f Analytical Chemistry, Vol. 71, No. 24, December 15, 1999
5485
Table 2. Effects of Mobile-Phase Additives on Positive Ion ESI Sensitivities of Antiviral Agents
compd I (MH+)/Cc I (MH+)/C I (MH+)/C I (MH+)/C I (MNH4+)/C I (MH+)/C I (MNa+)/C I (MNa+)/C I (MH+)/C I (MNa+)/C I (MH+)/C I (MNH4+)/C
mobile-phase additive
vidarabine 5-iodo-2′,3′5-iodo-2′zidovudine (AZT) apparent acycloguanosine vidarabine monophosphate dideoxyuridine deoxyuridine trifluridine a b b b b b b (347.2, 28.8) (338.1, 29.6) (354.1, 28.2) (296.2, 33.8) (267.2, 37.4)b pH (225.2, 44.4) (267.2, 37.4)
none 0.1% TFAH 1.0% HOAc 50 mM NH4OAc
6.4 2.3 3.1 7.1
0.5 mM NaOAc 0.5 mM NaOAc 5.0 mM NaOAc 0.5 mM NaI 0.5 mM NaI 50 mM NH4OH
6.0 6.0 6.9 6.9 6.9 10.1
0.12 0.14 0.59 0.17 0.01 0.12 0.17 0.13 0.06 0.25 0.31 0.03
0.13 0.18 0.70 0.18
