Analysis of Nucleic Acids by Capillary Ion-Pair Reversed-Phase

Analysis of Oligonucleotides by Liquid Chromatography and Mass Spectrometry. Soheil Pourshahian , Sean McCarthy. 2011,137-166 ...
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Anal. Chem. 1999, 71, 3730-3739

Analysis of Nucleic Acids by Capillary Ion-Pair Reversed-Phase HPLC Coupled to Negative-Ion Electrospray Ionization Mass Spectrometry Christian G. Huber* and Alexander Krajete

Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innrain 52a, A-6020 Innsbruck, Austria

Ion-pair reversed-phase high-performance liquid chromatography was successfully coupled to negative-ion electrospray ionization mass spectrometry by using 60 × 0.20 mm i.d. capillary columns packed with 2.3-µm micropellicular, octadecylated poly(styrene/divinylbenzene) particles as stationary phase and gradients of acetonitrile in 50 mM aqueous triethylammonium bicarbonate as mobile phase. Systematic variation of the eluent composition, such as concentration of ion-pair reagent, anion in the ion-pair reagent, solution pH, and acetonitrile concentration led to the conclusion that most parameters have opposite effects on chromatographic and mass spectrometric performances. The use of acetonitrile as sheath liquid enabled the rapid and highly efficient separation and detection of phosphorylated and nonphosphorylated oligonucleotides ranging in size from 8 to 40 nucleotides. High-quality full-scan mass spectra showing little cation adduction were acquired from which the molecular masses of the separated oligonucleotides were calculated with an accuracy of 0.011%. With calibration curves being linear over at least 2 orders of magnitude, the lower limits of detection for a oligodeoxythymidine 16mer were 104 fmol with full scan and 710 amol with selected-ion-monitoring data acquisition. The potential of ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was demonstrated for mixed-sequence oligomers by the characterization of a reaction mixture from solid-phase synthesis of a 40-mer oligonucleotide. The numerous problems posed by modern biochemistry, biology, and medicine as well as the growing significance of genetic engineering require the development of fast and reliable methods of utmost sensitivity and selectivity for the analysis of nucleic acids. Liquid-phase separation techniques, such as liquid chromatography1-5 and electrophoresis,6-9 have always played a * Corresponding author: (tel) +43 512 507 5176; (fax) +43 512 507 2767; (e-mail) [email protected]. (1) Hecker, R.; Riesner, D. J. Chromatogr. 1987, 418, 97-114. (2) Thompson, J. A.; Wells, R. D. Nature 1988, 334, 87-88. (3) Kasai, K.-I. J. Chromatogr. 1993, 618, 203-221. (4) Huber, C. G.; Stimpfl, E.; Oefner, P. J.; Bonn, G. K. LC-GC 1996, 14, 114127. (5) Huber, C. G. J. Chromatogr., A 1998, 806, 3-30. (6) Southern, E. M. Anal. Biochem. 1979, 100, 319-323. (7) Paulus, A.; Gassmann, E.; Field, M. J. Electrophoresis 1990, 11, 113-123.

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key role in the separation and detection of nucleic acids. During the past decade, mass spectrometry (MS) has become another important tool in the analysis of nucleic acids,10-12 mainly as a consequence of the introduction of electrospray ionization (ESI)13 and matrix-assisted laser desorption/ionization14 as very soft ionization mechanisms for large biomolecules. Moreover, miniaturized separation techniques, especially capillary high-performance liquid chromatography (capillary HPLC)15 and capillary electrophoresis,16 have had a profound impact on the modern practice of analyzing minute amounts of biopolymers contained in highly heterogeneous mixtures of biological origin. The flow rates in the microliter and submicroliter per minute range characteristic for capillary HPLC make this separation technique ideally suited for direct conjugation to ESI-MS. However, the detection and characterization of nucleic acids by ESI-MS is associated with difficulties arising from their polyanionic nature and tendency to form quite stable adducts with cations resulting in mass spectra of poor quality. Hence, removal of cations by off-line17-19 or on-line sample preparation techniques20-22 is obligatory in order to obtain high-quality mass spectra. Separation by HPLC is very useful for sample preparation of nucleic acids because it not only enables removal of cations from nucleic acid samples but also fractionates nucleic acids in more complex samples that cannot be directly analyzed by ESI(8) Schomburg, G. In Capillary Electrophoresis Technology; Guzman, N. A., Ed.; Chromatographic Science Series Vol. 64; Marcel Dekker Inc.: New York, 1993; pp 311-355. (9) Guttman, A.; Ulfelder, K. J. Adv. Chromatogr. 1998, 38, 301-340. (10) Bleicher, K.; Bayer, E. Biol. Mass Spectrom. 1994, 23, 320-322. (11) Portier, N.; Van Dorsselaer, A.; Cordier, Y.; Roch, O.; Bischoff, R. Nucleic Acids Res. 1994, 22, 3895-3903. (12) Little, D. P.; Thannhauser, T. W.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 95, 2318-2322. (13) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675-679. (14) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (15) Hirata, Y.; Novotny, M. J. Chromatogr. 1979, 186, 521-528. (16) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218, 209-216. (17) Stults, J. T.; Marsters, J. C. Rapid Commun. Mass Spectrom. 1991, 5, 359363. (18) Cheng, X.; Gale, D.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1995, 67, 586-593. (19) Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1994, 116, 4893-4897. (20) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (21) Liu, C.; Wu, Q.; Harms, A. C.; Smith, R. D. Anal. Chem. 1996, 68, 32953299. (22) Huber, C. G.; Buchmeiser, M. R. Anal. Chem. 1998, 70, 5288-5295. 10.1021/ac990378j CCC: $18.00

© 1999 American Chemical Society Published on Web 07/27/1999

MS. Anion-exchange HPLC and ion-pair reversed-phase HPLC (IPRP-HPLC) are the most popular chromatographic modes for the separation of nucleic acids.5 Whereas the salt gradients applied to elute the nucleic acids from anion-exchange columns are incompatible with ESI-MS, ion-pair reversed-phase chromatographic phase systems that employ volatile eluent components can be directly coupled to ESI-MS. Nevertheless, it is important to consider solution chemistry to achieve the best separation together with maximum analyte detectability, which usually implies that HPLC separation conditions have to be adjusted to be suitable for ESI-MS analysis. Usually, a compromise has to be found between optimum chromatographic and mass spectrometric conditions. Bleicher and Bayer chromatographed oligonucleotides containing up to 24 nucleotides on a 100 × 2 mm i.d. Nucleosil C18 column with gradients of acetonitrile in 10 mM ammonium acetate and detected the separated species by ESI-MS.23 An eluent containing 200 mM diisopropylammonium acetate in acetonitrilewater was used by Bothner et al. to separate oligonucleotides on a 150 × 0.5 mm i.d. column packed with a polymeric PLRP-S 5-µm 100-Å stationary phase. Phosphodiester, methylphosphonate, and phosphorothioate oligonucleotides up to 20-mers were characterized at the 7-nmol level with ESI-MS detection.24 Apffel et al. utilized a 250 × 2.1 mm i.d. YMC ODS-AQ C18 column to analyze oligonucleotides containing up to 75 nucleotides by HPLC-ESIMS.25,26 They found that 100 mM triethylammonium acetate, which is the optimal mobile-phase additive for IP-RP-HPLC of nucleic acids, resulted in drastic reduction of ion formation during ESI. Consequently, 1,1,1,3,3,3-hexafluoro-2-propanol was recommended as a volatile substitute for acetic acid as the acidic component of the ion-pair reagent, which resulted in efficient HPLC separation and analyte detectabilities in the low-picomole range. Nevertheless, compared to triethylammonium acetate, separation efficiency for larger oligomers was impaired with eluents containing triethylammonium hexafluoro-2-propanolate. We have synthesized and applied a micropellicular, octadecylated poly(styrene/divinylbenzene) stationary phase (PS/DVBC18) for high-resolution separation of oligonucleotides4,27,28 and DNA fragments.29-32 In this paper, we report on the feasibility of coupling this highly efficient chromatographic separation system to ESI-MS. The influence of solution chemistry on chromatographic and mass spectrometric performance is studied using different ion-pair reagents and oligonucleotides of different length and base composition. Several organic solvents are utilized as (23) Bleicher, K.; Bayer, E. Chromatographia 1994, 39, 405-408. (24) Bothner, B.; Chatmann, K.; Siuzdak, G. Bioorg. Med. Chem. Lett. 1995, 5, 2863-2868. (25) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. J. Chromatogr., A 1997, 777, 3-21. (26) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Anal. Chem. 1997, 69, 1320-1325. (27) Huber, C. G.; Oefner, P. J.; Bonn, G. K. Anal. Biochem. 1993, 212, 351358. (28) Oefner, P. J.; Huber, C. G.; Umlauft, F.; Berti, G. N.; Stimpfl, E.; Bonn, G. K. Anal. Biochem. 1994, 223, 39-46. (29) Huber, C. G.; Oefner, P. J.; Preuss, E.; Bonn, G. K. Nucleic Acids Res. 1993, 21, 1061-1066. (30) Huber, C. G.; Oefner, P. J.; Bonn, G. K. Chromatographia 1993, 37, 653658. (31) Oefner, P. J.; Huber, C. G.; Puchhammer-Sto ¨ckl, E.; Umlauft, F.; Bonn, G. K.; Kunz, C. BioTechniques 1994, 16, 898-908. (32) Huber, C. G.; Oefner, P. J.; Bonn, G. K. Anal. Chem. 1995, 67, 578-585.

sheath liquids to improve the stability of the electrospray and to enhance signal intensity in ESI-MS. Finally, the optimized IPHPLC-ESI-MS system is used to analyze synthetic 8-40-mer oligonucleotides. EXPERIMENTAL SECTION Chemicals and Oligonucleotide Samples. Acetonitrile (HPLC gradient grade), methanol (HPLC gradient grade), 2-propanol (analytical reagent grade), tetrahydrofuran (analytical reagent grade), and hydrochloric acid (37%, analytical reagent grade) were obtained from Merck (Darmstadt, Germany). Acetic acid (analytical reagent grade), formic acid (90%, analytical reagent grade), triethylamine (analytical reagent grade), and 1,1,1,3,3,3-hexafluoro2-propanol (HFIP, analytical reagent grade) were purchased from Fluka (Buchs, Switzerland). Solutions of the ion-pair reagents (triethylammonium acetate, TEAA; triethylammonium formate, TEAF; triethylammonium chloride, TEACl) were prepared by titrating aqueous solutions of triethylamine with the appropriate acid (acetic acid, formic acid, hydrochloric acid) until the desired pH was reached. A 1.0 M stock solution of triethylammonium bicarbonate (TEAB), pH 8.4-8.9, was prepared by passing carbon dioxide gas (AGA, Vienna, Austria) into a 1.0 M aqueous solution of triethylamine at 5 °C until the desired pH was reached. A 400 mM solution of triethylammonium hexafluoro-2-propanolate, pH 6.90, was prepared by titration of 400 mM hexafluoro-2-propanol with a 5.0% solution of triethylamine in water. For preparation of all aqueous solutions, high-purity water (Epure, Barnstead Co., Newton, MA) was used. The pH of the eluents was adjusted with an accuracy of (0.05 unit, and the given pH values always correspond to the neat aqueous solutions before addition of organic solvent. The standards of phosphorylated and nonphosphorylated oligonucleotides (p(dA)12-18, (dT)12-18, p(dT)12-18, p(dT)19-24, p(dT)25-30, (dT)16) were purchased as sodium salts from Pharmacia (Uppsala, Sweden) or Sigma-Aldrich (St. Louis, MO). The synthetic oligonucleotides (dT)16 (4805.15 Da), (dT)24 (7238.71 Da), and a 40-mer of mixed sequence (12 366.18 Da) were ordered from Microsynth (Balgach, Switzerland) and used without further purification. The average molecular mass of oligonucleotides was calculated using the following formula: molecular mass ) number of A’s × 312.207 + number of C’s × 288.177 + number of G’s × 328.207 + number of T’s × 303.187 - 61.965 + number of nucleotides × 1.008 Da. For phosphorylated oligonucleotides, 79.980 was added. Preparation of Packed Capillary Columns. Polyimidecoated fused-silica capillary tubing of 350-µm o.d. and 200-µm i.d. was obtained from Polymicro Technologies (Phoenix, AZ). A retaining frit was made at the end of a 15-cm-long fused-silica capillary by sintering a thin slug of 2-µm nonporous silica particles (Glycotech, Hamden, CT) wetted with a small droplet of sodium silicate solution (Sigma-Aldrich) by means of an electrically heated nickel-chrome filament ring. A slurry reservoir of 50-mm length, 6.35-mm o.d., and 2.45-mm i.d. and having an internal volume of 236 µL was fabricated from stainless steel tubing (Alltech, Deerfield, IL) and a pair of column end fittings (Valco Instruments Co. Inc., Houston, TX), with the holes of the fittings drilled to a diameter of 0.50 mm. The open end of the capillary was connected to the slurry reservoir with approximately 2 mm of the capillary protruding into the reservoir. Octadecylated poly(styrene/divinylbenzene) particles (PS/DVB-C18, 2.3 µm) were prepared Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

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according to the previously published protocol.27 Five milligrams of the PS/DVB-C18 stationary phase were suspended in 260 µL of tetrahydrofuran, sonicated for 10 min, and transferred to the slurry reservoir. The column was packed with methanol at a constant pressure of 70 MPa using an air-driven packing pump (Knauer, Berlin, Germany) until the length of the packed section in the column was 70-80 mm which took approximately 20 min. Then, the flow of the packing solvent was stopped, and after slowly releasing the pressure, the column was flushed with bidistilled water at 70 Mpa for 90 min before it was removed from the slurry reservoir and trimmed to a length of 60-70 mm. No frit was made at the inlet end of the capillary column. High-Performance Liquid Chromatography. The HPLC system consisted of a low-pressure gradient micropump (model Rheos 2000, Flux Instruments, Karlskoga, Sweden) controlled by a personal computer, a vacuum degasser (Knauer, Berlin, Germany), a column thermostat made from 3.3-mm-o.d. copper tubing, which was heated by means of a circulating water bath (model K 20 KP, Lauda, Lauda-Ko¨nigshofen, Germany), a microinjector (model C4-1004-0.02, Valco Instruments Co. Inc.) with a 20-nL internal sample loop, a variable-wavelength detector (model Linear UV-Vis 200, Linear Instruments, Fremont, CA) with a capillary detector cell (Grom, Herrenberg, Germany), and a PC-based data system (GynkoSoft, Version 5.22, Gynkotek, Germering, Germany). Electrospray Ionization Mass Spectrometry and Coupling with Capillary Liquid Chromatography. ESI-MS was performed on a Finnigan MAT LCQ quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an electrospray ion source. A syringe pump equipped with a 500-, 250-, or 100-µL glass syringe (Unimetrics, Shorewood, IL) was used for continuous infusion experiments and for pumping sheath liquid. For analysis with pneumatically assisted ESI, an electrospray voltage of 3.74.0 kV and a nitrogen sheath gas flow of 60-80 arbitrary units were employed. The temperature of the heated capillary was set to 200 °C. Total ion chromatograms and mass spectra were recorded on a personal computer with the LCQ Navigator software version 1.2 (Finnigan). Mass calibration and coarse tuning was performed in the positive-ion mode by direct infusion of a solution of caffeine (Sigma), methionyl-arginyl-phenylalanyl-alanine (Finnigan), and Ultramark 1621 (Finnigan). Fine-tuning for ESI-MS of oligonucleotides in the negative-ion mode was performed with a 0.36 mg/mL solution of (dT)24 (Microsynth) in 10 mM triethylamine-50% water-50% acetonitrile (v/v). For all direct infusion experiments, cations present in the oligonucleotide samples were removed by on-line cation exchange using a 20 × 0.50 mm i.d. cation-exchange microcolumn packed with 38-75-µm Dowex 50 WX8 particles (Serva, Heidelberg, Germany).22 For IP-HPLCESI-MS analysis, oligonucleotides were injected without prior cation removal. RESULTS AND DISCUSSION Separation of Oligonucleotides in 200-µm-i.d. Capillary Columns. IP-RP-HPLC is a well-established technique for separating oligonucleotides and double-stranded DNA, and a variety of silica- and polymer-based packing materials are commercially 3732 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

Figure 1. High-resolution capillary HPLC separation of phosphorylated oligonucleotide ladders: column, PS/DVB-C18, 2.1 µm, 70 × 0.20 mm i.d.; mobile phase, (A) 100 mM TEAA, pH 7.00, (B) 100 mM TEAA, pH 7.00, 20% acetonitrile; linear gradient, 15-45% B in 3.5 min, 45-55% B in 2.5 min, 55-59% B in 4.0 min; flow rate, 3.3 µL/min; temperature, 50 °C; detection, UV, 254 nm; sample, p(dA)12-18, 1.0 ng each, p(dT)12-30, 0.70 ng each.

available.5,33 One of the unique properties of micropellicular PS/ DVB-C18 is the capability of this stationary phase to separate nucleic acids with high resolution, which has been demonstrated by the separation of oligodeoxyadenylic acids with chain lengths of 10-90 nucleotides with single-base resolution in less than 30 min.4 However, to date, no capillary columns packed with PS/ DVB-C18 have been employed, mainly because it is difficult to pack the small polymeric particles efficiently into fused-silica capillaries and because reproducible fabrication of a permeable and stable frit at the column outlet is tedious. We manufactured a very thin frit at the column outlet by sintering nonporous silica particles with the help of an electrically heated filament ring. Before sintering, the particles were wetted with sodium silicate solution to aid in sticking the particles together. Subsequently, the PS/DVB-C18 particles could be packed into a 0.20-mm-i.d. fused-silica capillary using pressures up to 75 MPa without damage to the frit. Figure 1 illustrates the separation of phosphorylated oligodeoxyadenylic and oligodeoxythymidylic acids, ranging in size from 12 to 30 nucleotides, on a 70 × 0.20 mm PS/DVB-C18 capillary column. Applying a gradient of 3.0-9.0% acetonitrile in 100 mM triethylammonium acetate, all 26 oligonucleotides were well resolved within 9.0 min. The peak widths at half-height of the eluted oligonucleotides ranged from 2.8 s for p(dT)13 to 5.3 s for p(dT)30, which indicates the high efficiency and peak capacity of this chromatographic system. Moreover, this separation clearly demonstrates that oligonucleotide separations are feasible with 0.20-mm-i.d. capillary columns and with the commonly used 4.6mm-i.d. columns with equivalent separation performance (compare, e.g., Figure 4 in ref 27). To render this chromatographic separation system ESI-MS compatible, several factors have to be considered that are related to the solution chemistry of the column effluent to be electrosprayed. Bleicher and Bayer23 as well as Apffel et al.25 reported that 100 mM triethylammonium acetate is not suitable as an ionpair reagent for HPLC-ESI-MS because of the poor detectability of the eluted oligonucleotides by ESI-MS. Reducing the concentra(33) Zon, G. In High-Performance Liquid Chromatography in Biotechnology; Hancock, W., Ed.; Wiley: New York, 1990.

tion of triethylammonium acetate improved the signal intensity in ESI-MS but, on the other hand, significantly deteriorated the chromatographic separation performance. The strong signalsuppressing effect of triethylammonium acetate was attributed to the preferential evaporation of triethylamine from the electrosprayed microdroplets leaving an excess of acetic acid in the droplets which reduces ionization efficiency of the dissolved oligonucleotides.25 Moreover, the concentration of organic solvent in the electrosprayed solution was shown to have a significant impact on ionization efficiency. An increase in the acetonitrile concentration in the electrosprayed solution from 0 to 80% resulted in a more than 25-fold increase in signal intensity.10 Charge-state distribution and ionization efficiency are also affected by solution pH. In general, higher charge states, a higher number of charge states, and higher signal intensities were observed when oligonucleotides were electrosprayed from solutions of high pH.10,18 To find the best compromise for the analysis of oligonucleotides by IP-RP-HPLC-ESI-MS, the influence of the following solution parameters on chromatographic and mass spectrometric performance were investigated independently using IP-RP-HPLC and ESI-MS: type of anion in the ion-pair reagent, solution pH, and concentration of ion-pair reagent. Additionally, the feasibility of applying a sheath liquid to match chromatographic and mass spectrometric conditions was studied. Influence of Mobile-Phase Composition on Chromatographic Performance. In IP-RP-HPLC, the chromatographic phase system comprises a hydrophobic stationary phase and a hydroorganic eluent containing an amphiphilic ion and a hydrophilic counterion. Retention of oligonucleotides is determined by several factors such as hydrophobicity of the stationary phase, charge, hydrophobicity and concentration of the amphiphile, ionic strength and dielectric constant of the mobile phase, and concentration of organic modifier. According to the electrostatic model,34 retention is effected by the electrostatic interactions between the positive surface potential generated by the amphiphilic ions adsorbed at the stationary phase, triethylammonium ions, for instance, and the negative surface potential generated by the dissociated phosphodiester groups of the oligonucleotides. As a consequence of increasing the concentration of an organic modifier such as acetonitrile, amphiphilic ions are desorbed from the stationary phase, resulting in elution of the retained analytes. The influence of triethylammonium bicarbonate concentration in the eluent on retention and resolution of oligonucleotides is shown in Figure 2. To measure the retention times and resolution of oligonucleotides, a (dT)12-18 standard was eluted with a gradient of 5-10% acetonitrile in 25-100 mM triethylammonium bicarbonate in 15 min. Between 25 and 75 mM triethylammonium bicarbonate, retention times increased with increasing concentration of the ion-pair reagent because of the higher surface potential at the stationary phase (Figure 2a). A slight decrease in retention is observed with 100 mM triethylammonium bicarbonate, which reflects the limited adsorption capacity of the stationary phase for the amphiphile35 and the effect of ionic strength on the surface potentials. Resolution of oligonucleotides gradually improved with increasing ion-pair reagent concentration as depicted in Figure 2b. On average, resolution of (dT)12-18 was 12% better with 100 (34) Bartha, A.; Stahlberg, J. J. Chromatogr., A 1994, 668, 255. (35) Jost, W.; Unger, K.; Schill, G. Anal. Biochem. 1982, 119, 214-223.

Figure 2. Influence of ion-pair reagent concentration on retention (a) and resolution (b) in IP-RP-HPLC of oligonucleotides: column, PS/DVB-C18, 2.1 µm, 60 × 4.0 mm i.d.; mobile phase, (A) 100 mM TEAB, pH 7.50, (B) 100 mM TEAB, pH 7.50, 10% acetonitrile; linear gradient, 50-100% B in 15 min; flow rate, 550 µL/min; temperature, 50 °C; detection, UV, 254 nm; sample, (dT)12-18, 36 ng each.

mM triethylammonium bicarbonate compared to 25 mM triethylammonium bicarbonate. Since triethylammonium ion does not carry a permanent positive charge, an acidic compound such as acetic acid is usually added to the mobile phase to protonate the amine. As reported in the literature, acids of higher volatility should offer better detectability for ESI-MS.25 Therefore, formic acid, hydrochloric acid, and carbon dioxide were employed as more volatile acidic components of the ion-pair reagent. The different counterions in the ion-pair reagent had only a moderate effect on retention times and chromatographic separation efficiency. Applying a gradient of 5.0-12% acetonitrile in 12 min in 100 mM solutions of the ionpair reagents (other conditions as in Figure 1), retention times slightly increased in the sequence triethylammonium acetate (3.78 min for p(dT)12) ≈ triethylammonium bicarbonate (3.88 min for p(dT)12) < triethylammonium chloride (4.21 min for p(dT)12) < triethylammonium formate (4.46 min for p(dT)12) and the maximum and minimum retention times for p(dT)30, for example, differed by 11%. The resolution between p(dT)12 and p(dT)13 was highest with triethylammonium bicarbonate (R ) 4.52) and lowest with triethylammonium acetate (R ) 3.72) with resolution values Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

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differing by 18%. However, the separation efficiency for longer oligonucleotides such as p(dT)29 and p(dT)30 was similar with all four ion-pair reagents, and the observed differences in resolution were all within the experimental error (R ) 1.05-1.27). The effect of eluent pH on retention and resolution of oligonucleotides was studied in a pH range of 6.80-10.40. As the pH of solutions containing triethylammonium bicarbonate can change because of evaporation of carbon dioxide, this study was performed using triethylammonium acetate as ion-pair reagent. From pH 6.80-8.50, the pH dependence of retention is characterized by a small decrease in retention times, e.g., from 4.0 to 3.6 min for p(dT)12 (5-12% acetonitrile in 100 mM TEAA, pH 6.8010.5, in 12 min, 2.3 µL/min; other conditions as in Figure 1). At pH values higher than 8.50, retention times rapidly decreased due to the beginning deprotonation of triethylammonium ion, and at pH 10.40, all oligonucleotides eluted in the void volume. Stronger retention did not entail better resolution of oligonucleotides because the dependence of resolution on pH showed a maximum at pH 8.50. Influence of Solvent Composition on Mass Spectrometric Performance. Solvent properties influence electrospray characteristics in a variety of ways, notably the dependency of the minimal potential required to form the Taylor cone on surface tension, the spray current on conductivity, the droplet size on viscosity, and the charge-state distribution on dielectric constant and pH.36 For direct infusion analysis, oligonucleotides are commonly introduced into the mass spectrometer in solutions containing a 10-50 mM concentration of an organic base and an organic modifier, typically 50% or greater, with higher concentrations giving higher signal intensity.10,18,37 Since oligonucleotides are very polar compounds and require high desolvation energies to form gas-phase ions, increased signal intensity at higher concentrations of organic modifier is mainly a consequence of lower surface tension and faster evaporation of solvent from the electrosprayed droplets. Unfortunately, the concentration of organic modifier cannot be chosen independently in IP-RP-HPLC because the solvent strength has to be adjusted to allow chromatographic retention of the analytes (compare Figure 5 in ref 27). Figure 3a illustrates the decrease in signal intensity when the acetonitrile concentration optimal for ESI-MS is reduced to a concentration practicable for IP-RP-HPLC. With a spray solvent containing 10 mM triethylamine and 50% acetonitrile, a bimodal charge-state distribution is observed, with the 3- and the 5charge states being the local maximums. Upon changing the acetonitrile concentration from 50 to 10%, the total ion current (TIC, calculated as the sum of the peak heights of all individual charge states) is reduced by a factor of 4.0 (243 500 vs 969 580 counts) whereas the charge-state distribution is not significantly affected. As mentioned above, a solution of neat triethylamine does not work as an ion-pair reagent for IP-RP-HPLC due to the lack of a permanent positive charge in the amphiphile. Protonation of triethylamine with an acid has two major effects on solution properties. First, the pH of the solution decreases (for a 10 mM (36) Wang, G.; Cole, R. B. In Electrospray Ionization Mass Spectrometry Fundamentals, Instrumentation and Applications; Cole, R. B., Ed.; John Wiley and Sons: New York, 1997; pp 137-174. (37) Greig, M. J.; Griffey, R. H. Rapid Commun. Mass Spectrom. 1995, 9, 97102.

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Figure 3. Influence of acetonitrile concentration (a) and ion-pair reagent concentration (b) on signal intensity and charge-state distribution in ESI-MS of oligonucleotides: cation-exchange microcolumn, Dowex 50 WX8, 20 × 0.50 mm; scan, 200-2500 amu; electrospray voltage, 4.0 kV; sheath gas, 75 units; direct infusion of 0.24 mg/mL (dT)16 in (a) 10 mM triethylamine, pH 11.30, 10% and 50% acetonitrile, respectively, and (b) 10 mM and 50 mM TEAB, pH 8.90, respectively, 10% acetonitrile; flow rate, 3.0 µL/min.

triethylamine solution from pH 11.30 to pH 7.00-9.00, depending on the amount of acid added), and second, the conductivity of the solution increases. The effect of titration of 10 mM triethylamine with carbon dioxide to pH 8.90 on MS performance can be deduced from Figure 3b. Compared to 10 mM triethylamine (Figure 3a), the total ion current decreases by a factor of 3.0 (81 695 vs 243 500 counts). Moreover, upon addition of the acid, the 3- charge state becomes the predominant charge state as a result of charge-state reduction.18,38 The effect of acids on chargestate distribution can be explained on the basis of solution and gas-phase acid-base equilibria. The more acidic the solution, the more likely acids will donate protons to oligonucleotide anions and reduce the charge states of oligonucleotides. An increase in the triethylammonium bicarbonate concentration from 10 to 50 mM, which is desirable to obtain better chromatographic performance, entailed a further decrease in signal intensity by a factor of 3.0 (27 377 vs 81 695 counts, Figure 3b). In summary, the adaptation of conditions optimal for ESI-MS (10 mM triethylamine in 50% water-50% acetonitrile) to conditions suitable for chromatographic separations (50 mM triethylammonium bicarbonate in 90% water-10% acetonitrile) was accompanied by a 35-fold decrease in mass signal intensity. (38) Muddiman, D. C.; Cheng, X.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 797-706.

Table 1. Properties of Anions in the Ion-Pair Reagent and Their Influence on Signal Intensity in ESI-MS of Oligonucleotides counterion in the ion-pair reagent acetate bicarbonate formate chloride

Figure 4. Influence of type of ion-pair reagent on signal intensity and charge-state distribution in ESI-MS of oligonucleotides: cationexchange microcolumn, Dowex 50 WX8, 20 × 0.50 mm; scan, 2002500 amu; electrospray voltage, 4.0 kV; sheath gas, 75 units; direct infusion of 0.24 mg/mL (dT)16 in 50 mM TEAA, TEAB, TEAF, or TEACl, pH 8.90, 10% acetonitrile; flow rate, 3.0 µL/min.

The effect of counterion in the ion-pair reagent on ESI-MS performance was investigated in a series of experiments where a 0.24 mg/mL solution of (dT)16 was electrosprayed from solutions containing 10% acetonitrile and 50 mM triethylamine which has been titrated to pH 8.90 with acetic acid, carbon dioxide, formic acid, or hydrochloric acid. The signal intensities of the individual charge states and the total ion current decreased with the different counterions in the order acetate > bicarbonate > formate > chloride (Figure 4a). Under otherwise identical conditions, the exchange of acetate with chloride resulted in a more than 5-fold decrease in total ion current (7144 vs 37 073 counts). This finding contradicts the thesis that the volatility of the counterion in the ion-pair reagent has a strong influence on signal intensity in ESIMS of oligonucleotides because the most intense signal is observed with the acid of highest boiling point (Table 1). The suppression of oligonucleotide signal upon addition of the acids is most likely due to the competition of anions for ionization. Since ESI generates a roughly constant ion current, an increase in the intensity from added acids will reduce the intensity of the oligonucleotide ions. Moreover, ions of higher conductivity will be more efficient in signal suppression. As shown in a semilogarithmic plot of signal intensity vs limit equivalent conductivity, there is a clear correlation between conductivity and MS detect-

boiling point of conjugated acid (K)

limit equiv conductivity of anion (cm2 S mol-1)

TIC (counts)

391 195 (sublimation) 374 188

40.9 44.5 54.6 76.4

37 073 27 378 21 010 7 144

Figure 5. Influence of pH on signal intensity and charge-state distribution in ESI-MS of oligonucleotides: cation-exchange microcolumn, Dowex 50 WX8, 20 × 0.50 mm; scan, 200-2500 amu; electrospray voltage, 3.7 kV; sheath gas, 63-70 units; direct infusion of 0.24 mg/mL (dT)16 in 50 mM TEAA, pH 6.80-9.90, 10% acetonitrile; flow rate 3.0 µL/min; sheath liquid, acetonitrile; flow rate, 3.0 µL/min.

ability (Figure 4b), the anion of highest limit equivalent conductivity (chloride) being the most efficient in signal suppression. The effect of solution pH on signal intensity and charge-state distribution was studied by variation of the pH from 6.8 to 9.90 through addition of appropriate amounts of acetic acid. Figure 5 shows that the pH of the electrosprayed solution had no significant effect on signal intensity and charge-state distribution of multiply charged ions of (dT)16 in the pH range 6.80-8.40 that is utilizable for IP-RP-HPLC. However, a significant improvement in signal intensities was observed at pH values higher than 9.00, which are, nevertheless, not practicable as chromatographic separation conditions. This finding suggests that the effect of pH on signal intensity is mainly due to the higher amount of acetic acid needed to titrate the triethylamine to lower pH. The concentrations of acetic acid necessary to titrate 50 mM triethylamine to pH 6.80, 8.40, and 9.90 were 50.4, 49.9, and 47.0 mM, respectively. Because the titration curve of triethylamine with acetic acid has its greatest slope between pH 6.00 and 8.50, the difference in acetic acid concentration is very small in this pH range (0.50 mM difference between pH 6.80 and 8.40) and the signal intensities were almost constant (Figure 5). However, between pH 8.40 and 9.90 the difference is 2.9 mM, representing a 5.8% reduction in acetic acid concentration, which resulted in a 48% increase in total ion current due to the lower conductivity of the solution (722 668 vs 486 729 counts). Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

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Figure 6. Comparison of different solvents for ESI-MS of oligonucleotides without and with addition of sheath liquid: cationexchange microcolumn, Dowex 50 WX8, 20 × 0.50 mm; scan, 2002500 amu; electrospray voltage, 3.7 kV; sheath gas, 60-70 units; direct infusion 0.24 mg/mL (dT)16 in 50 mM TEAB, pH 8.40, 10% acetonitrile (a); 50 mM TEAB, pH 8.40, 10% acetonitrile (b); 10 mM triethylamine, pH 11.30, 50% acetonitrile (c); 2.2 mM triethylamine, 400 mM HFIP, pH 6.90, 20% methanol (d); flow rate 3.0 µL/min; no sheath liquid in (a, c, d); sheath liquid, acetonitrile; flow rate, 3.0 µL/ min in (b).

Addition of Sheath Liquid To Improve the MS Signal with IP-RP-HPLC-Compatible Solvents. From the results discussed in the preceding section, it follows that the major factors responsible for the relatively low intensity of oligonucleotide signals with eluents containing triethylammonium acetate and 10% acetonitrile are high surface tension because of the low concentration of organic solvent and high conductivity because of addition of the acid. The addition of an organic solvent as sheath liquid through the triaxial electrospray probe was supposed to alleviate these problems because it reduces the surface tension and dilutes the ion-pair reagent in the electrosprayed droplets. This concept of adding a sheath liquid to the column effluent is more frequently used for the coupling of capillary electrophoresis with ESI-MS, which permits the independent optimization of the electrolyte composition for capillary electrophoresis and ESI-MS.39,40 A variety of different sheath liquids including methanol, 2-propanol, acetonitrile, hexafluoro-2-propanol, triethylamine, 10 mM triethylamine in acetonitrile, and 400 mM hexafluoro-2propanol in methanol were tested, acetonitrile being the most efficient in improving signal intensity. Figure 6 depicts the signal intensities of multiply charged ions of (dT)16 obtained with different spray solvents without and with the addition of sheath liquid. Comparison of Figure 6a and b reveals that the addition of 3.0 µL/min acetonitrile as sheath liquid to a solution of the oligonucleotide in 50 mM triethylammonium bicarbonate-10% acetonitrile brought about a 6.6-fold increase in the signal intensity of the 3- charge state of (dT)16. Due to charge-state reduction, the 2- and 3- charge states were the most abundant, although small signals for the 4-, 5-, and 6- charge states were also present. The total ion current increased from 83 881 to 652 604 (39) Smith, R. D.; Olivares, J. A.; Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988, 60, 436-441. (40) Moseley, M. A.; Jorgenson, J. W.; Shabanowitz, J.; Hunt, D. F.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 1992, 3, 281-288.

3736 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

Figure 7. IP-RP-HPLC-ESI-MS analysis of homologous oligonucleotides ranging in size from 12 to 30 nucleotides with triethylammonium hexafluoro-2-propanolate as ion-pair reagent: column, PS/DVB-C18, 2.1 µm, 60 × 0.20 mm i.d.; mobile phase, (A) 2.2 mM TEA, 400 mM HFIP, pH 6.90, (B) 2.2 mM TEA, 400 mM HFIP, pH 6.90, 40% methanol; linear gradient, 23-38% B in 4.0 min, 38-63% B in 12 min; flow rate, 2.5 µL/min; temperature, 50 °C; scan, 10003100 amu; electrospray voltage, 3.7 kV; sheath gas, 67 units; sample, p(dT)12-18, 0.57 ng each, p(dT)19-24, 1.33 ng each, p(dT)25-30, 1.33 ng each.

counts (factor 7.8) upon the addition of sheath liquid. Moreover, the signal intensity of the 3- charge state was equal to the signal intensity obtained with 10 mM triethylamine-50% acetonitrile, a typical carrier for direct infusion ESI-MS of oligonucleotides (Figure 6c). However, the number of charge states observed with the latter solvent was greater (2- to 11-), resulting in a notably higher total ion current (1 906 000 counts). The highest total ion current and a shift to lower charge states (10- to 2-) was obtained with a solvent containing 2.2 mM triethylamine-400 mM hexafluoro-2-propanol-20% methanol (2 226 975 counts, Figure 6d). Coupling of IP-RP-HPLC with ESI-MS. The analysis of a series of phosphorylated oligodeoxythymidylic acids ranging in size from 12 to 30 nucleotides by IP-RP-HPLC coupled to ESI-MS detection using the eluent system proposed by Apffel et al.25,26 and a 200-µm-i.d. capillary column packed with PS/DVB-C18 is shown in Figure 7. With an eluent containing 2.2 mM triethylamine-400 mM hexafluoro-2-propanol and a gradient of 9.2-15.2% methanol in 4.0 min, followed by a gradient of 15.2-25.2% in 12

min, all oligonucleotides were separated almost to baseline within 9.0 min (Figure 7a). The peak widths at half-height of p(dT)12 and p(dT)30 were 7.2 and 25.8 s, respectively. The reconstructed ion chromatogram (RIC) was characterized by a relatively high background level of 2.20 × 106 counts compared to the maximum signal intensity of p(dT)13 of 6.34 × 106 counts. A total of 155 fmol of p(dT)12 could be detected with a signal-to-noise-ratio of 6.4:1 whereas 145 fmol of p(dT)30 yielded a signal only slightly above the noise level. Panels b and c of Figure 7 depict the mass spectra obtained by averaging 15-20 scans under the peaks of p(dT)12 and p(dT)30. Extensive cation adduction is evident in the signals for the 2- charge state of p(dT)12. The investigation of the influence of eluent composition on chromatographic and mass spectrometric performance has revealed that most parameters have quite opposite effects: high acetonitrile concentration is optimal for ESI-MS detectability but not practicable for IP-RP-HPLC, high concentration of ion-pair reagent improves chromatographic separation but suppresses the signal in ESI-MS, and oligonucleotides are better ionized but not retained on the stationary phase at high pH. Nevertheless, acetonitrile added as sheath liquid to the column effluent helped to more closely match the demands of IP-HPLC and ESI-MS. Therefore, the following conditions were regarded to be a good compromise for IP-RP-HPLC-ESI-MS analysis of oligonucleotides: a gradient of 5.0-10% acetonitrile in 50 mM triethylammonium bicarbonate, pH 8.40, and addition of acetonitrile as sheath liquid. The separation of the p(dT)12-30 sample by applying the elaborated eluent system is shown in Figure 8. With a gradient of 5.0-7.0% acetonitrile in 2.0 min followed by 7.0-10% in 6.5 min in 50 mM triethylammonium bicarbonate, all 19 components of the standard were resolved within 5.5 min. The improved chromatographic performance with this eluent compared to triethylammonium hexafluoro-2-propanolate is reflected in peak widths at half-height of 3.6 and 6.6 s for p(dT)12 and p(dT)30, respectively. The background level with triethylammonium bicarbonate is significantly lower (1.52 × 105 counts) and the signalto-noise ratios for 256 fmol of p(dT)12 and 120 fmol of p(dT)30 were 12.5:1 and 3.8:1, respectively. High-quality mass spectra could be extracted from the reconstructed ion chromatogram allowing the unambiguous identification of the eluted oligonucleotides (see, for example, Figure 8b and c) by their molecular masses. An average relative mass deviation of 0.011% (N ) 19) between the measured masses and those calculated from the sequences of p(dT)12-30 clearly corroborates the high mass accuracy of IPHPLC-ESI-MS, which is necessary to identify compounds on the basis of their molecular masses. Adduction with alkali and alkaline earth metal ions has always been a major problem in negative-ion ESI-MS of oligonucleotides.10,41 Whereas on-line cation exchange enables the trapping of cations on a cation exchanger of relatively high affinity for the cations, removal of cations during IP-RP-HPLC has to take place in the mobile phase through competition of an excess of triethylammonium ions with metal cations for the negative charges at the sugar-phosphate backbone. Figure 9 shows the expanded views of the signals for the most abundant charge state of p(dT)15 using 50 mM triethylammonium bicarbonate (Figure 9a) and 2.2 (41) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882-889.

Figure 8. IP-RP-HPLC-ESI-MS analysis of homologous oligonucleotides ranging in size from 12 to 30 nucleotides with triethylammonium bicarbonate as ion-pair reagent: column, PS/DVB-C18, 2.1 µm, 60 × 0.20 mm i.d.; mobile phase, (A) 50 mM TEAB, pH 8.40, (B) 50 mM TEAB, pH 8.40, 20% acetonitrile; linear gradient, 2535% B in 2.0 min, 35-50% B in 6.5 min; flow rate, 2.9 µL/min; temperature, 50 °C; scan, 1000-3100 amu; electrospray voltage, 3.7 kV; sheath gas, 78 units; sheath liquid, acetonitrile; flow rate, 3.0 µL/ min; sample, p(dT)12-18, 0.94 ng each, p(dT)19-24, 1.1 ng each, p(dT)25-30, 1.1 ng each.

mM triethylamine-400 mM hexafluoro-2-propanol (Figure 9b) which have been extracted from the reconstructed ion chromatograms in Figure 8a and Figure 7a. It is evident that adduction of p(dT)15, which had been injected as the pentadecasodium salt, with sodium and potassium ions was efficiently reduced during IP-RP-HPLC with 50 mM triethylammonium bicarbonate as eluent. The intensities of the monosodium and monopotassium adducts amounted to 2.1 and 9.8%, respectively, of the total ion intensity of the 3- charge state. Although the sample has been injected as the sodium salt, the higher abundance of the monopotassium adduct indicates a higher affinity of potassium for the phosphodiester groups of the oligonucleotides. The presence of small amounts of sodium and potassium adducts was highly advantageous for the determination of the charge state of a signal, especially when only one charge state could be observed due to charge-state reduction with triethylammonium bicarbonate. With 2.2 mM triethylamine-400 mM hexafluoro-2-propanol, however, extensive cation adduction was observed with adducts Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

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Table 2. Identification of Compounds Found in the Reaction Mixture of a Synthetic 40-Mer Oligonucleotide mass (Da) peak no.

retention time (min)

m/za

charge stateb

calcd

theor

peak identificationc

1 2 3 4 5 6 7 8

1.82 2.98 3.05 3.26 4.25 4.51 5.44 5.44

2071.42 3018.04 3307.23 3596.41 4823.20 5769.83 5839.92 12366.18

7-mer 10-mer 11-mer 12-mer 16-mer 19-mer 19-mer + 1 isobutyryl group 40-mer target product

6.03

12433.0

12436.27

40-mer + 1 isobutyryl group

10

6.60

12503.0

12506.36

40-mer + 2 isobutyryl groups

11

7.18

222233354545454-

2070.8 3017.0 3305.6 3595.2 4821.3 5768.7 5838.0 12364.0

9

1034.4 1507.5 1651.8 1796.6 1606.1 1921.9 1945.0 2471.9 3089.6 2485.6 3107.1 2499.5 3125.0 2513.3 3142.1

12572.0

12576.45

40-mer + 3 isobutyryl groups

a m/z of the (M - nH)n- signal(s) observed in the extracted mass spectra. b The charge state was calculated from the mass difference between the (M - nH)n- and the (M - (n + 1)H + K)n- signals. c Sequence: TGA TGA TGA TGC GTG AAG ACA GTA GTT CCC TGA CTC TGA T. Since oligonucleotides are synthesized beginning at the 3′-end by the solid-phase method, the sequence of failure sequences has to be read from right to left.

Figure 9. Cation adduction of oligonucleotides with eluents containing 50 mM triethylammonium bicarbonate (a) and 2.2 mM triethylammonium hexafluoro-2-propanolate (b) as ion-pair reagent. Chromatographic conditions as in Figures 7 and 8.

ranging from the monosodium to the tetrasodium-monopotassium adduct (Figure 9b). Such adduction is undesirable because the signal intensity is distributed among many species resulting in a low signal-to-noise ratio for the detected species. Moreover, accurate mass determinations using the signals of higher charge states are hampered because of the overlapping of adduct signals. The intensity of all adduct signals was 72.7% of the total ion intensity of the 2- charge state leaving only 27.3% intensity for the (M - 2H)2- ion. The reason for the difference between triethylammonium bicarbonate and triethylammonium hexafluoro2-propanolate with respect to their efficiency in reducing cation adduction is most probably the difference in the concentration of triethylammonium ions. Eluents containing higher concentrations of triethylammonium ions are obviously more efficient in replacing cations at the sugar-phosphate backbone during IP-HPLC of oligonucleotides. 3738 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

Reproducibility, Detection Limits, and Quantitation. The reproducibility of peak areas and retention times was checked by repeated injections of (dT)16 (Pharmacia) using the mass spectrometer in the full-scan mode (1000-3100 amu). The peak areas and retention times of nine injections of 1.04 pmol of (dT)16 were reproducible with relative standard deviations of 0.85 and 9.0%, respectively, which both represent good values for qualitative and quantitative analysis. In the full-scan mode, a 16-mer was determined with a lower mass detection limit of 500 pg (104 fmol) at a signal-to-noise ratio of 3:1. The plot of peak areas vs injected amount showed good linearity (R2 ) 0.9982) over a range of more than 2 orders of magnitude (5.20 µM-1.04 mM (dT)16, 20-nL injection volume) with the equation of the linear regression line being Y ) 7639X - 1683 (Y, peak area, calculated as the average of three injections (arbitrary units); X, amount of injected oligonucleotide (pmol); N ) 6). In the selected-ion-monitoring mode, the 3- charge state of (dT)16 at m/z 1600.72 was detected with a lower limit of detection of 710 amol at a signal-to-noise ratio of 3:1, which represents an improvement in detectability by a factor of 146 compared to the full-scan mode. Application of IP-RP-HPLC to the Quality Control of Synthetic Oligonucleotides. With the increasing utilization of synthetic oligonucleotides in many areas of biochemistry and biotechnology as primers, probes, templates for the construction of mutations, and cloning adaptors, there is a growing need to characterize the identity and purity of the synthesized oligonucleotides. The major byproducts of a chemical oligonucleotide synthesis reaction using the solid-phase phosphoramidite method are failure sequences and partially deprotected target sequences. Figure 10 depicts the analysis of a crude 40-mer oligonucleotide after solid-phase synthesis by IP-RP-HPLC-ESI-MS. For this separation, a gradient of 2.0-12% acetonitrile in 50 mM triethylammonium bicarbonate in 10 min was applied. Extraction and analysis of the mass spectrum allowed the identification of the peak eluting at 5.44 min as the fully deprotected target sequence (Figure 10b). Coeluting with the target product was an isobutyrylprotected 19-mer failure sequence. Moreover, the chromatogram

the amino group of deoxyguanosine phosphoramidite during solidphase synthesis. The four most abundant failure sequences were the 10-, 11-, 12-, and 19-mer. Looking at the sequence of the target product, one finds that the nucleotide following at position 20 (counting from the 3′-end) is deoxyadenosine and the nucleotides following at positions 11, 12, and 13 are all deoxycytidines, which indicates that the efficiencies of some of the A- and C-coupling cycles were rather poor in this particular synthesis reaction.

Figure 10. Characterization of a synthetic 40-mer oligonucleotide raw product: column, PS/DVB-C18, 2.1 µm, 60 × 0.20 mm i.d.; mobile phase, (A) 50 mM TEAB, pH 8.40, (B) 50 mM TEAB, pH 8.40, 20% acetonitrile; linear gradient, 10-60% B in 10 min; flow rate, 2.9 µL/min; temperature, 50 °C; scan, 1000-3500 amu; electrospray voltage, 3.7 kV; sheath gas, 80 units; sheath liquid, acetonitrile; flow rate, 3.0 µL/min; sample, 48 ng of raw product. Peak identification in Table 2.

in Figure 10a showed nine additional peaks whose identity was readily established on the basis of their measured masses (Table 2). The three peaks following the target product were characterized by mass differences of 69.0, 2 × 69.5, and 3 × 69.7 Da relative to the target product and correspond the mass of one, two, or three isobutyryl protecting groups that have been used to protect

CONCLUSIONS It is concluded that the effects of solution parameters on the performance of IP-RP-HPLC and negative-ion ESI-MS are contrary: conditions ideal for one method are frequently nonideal for the other. Therefore, a compromise has to be found to enable the efficient on-line coupling of IP-HPLC to ESI-MS. If an organic sheath liquid is added to the column effluent, conditions commonly used for IP-HPLC of nucleic acids are compatible with ESIMS. Using capillary columns of 200-µm-i.d. packed with micropellicular PS/DVB-C18 particles and gradients of acetonitrile in 50 mM triethylammonium bicarbonate, oligonucleotides up to at least 40-mers can be separated within 10 min with singlenucleotide resolution. Cation adduction in the separated oligonucleotides is efficiently reduced, allowing highly accurate mass determinations. Detection limits in the lower femtomole range with full-scan mode and in the upper attomole range with selected-ion monitoring make the method attractive to obtain comprehensive information about low amounts of nucleic acids present in samples of synthetic and biological origin. ACKNOWLEDGMENT The authors are grateful to Csaba Horva´th (Yale University, New Haven, CT) for the gift of nonporous silica particles. This work was supported by a grant from the Austrian Science Fund (P13442). Received for review April 9, 1999. Accepted June 7, 1999. AC990378J

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