On-Column Electrochemical Reactions Accompanying the

Analytical Chemistry 2004 76 (22), 6659-6664 ... Study of electrochemical oxidation of cyanidin glycosides by online combination of electrochemistry w...
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Anal. Chem. 2003, 75, 1022-1030

On-Column Electrochemical Reactions Accompanying the Electrospray Process Suya Liu, William J. Griffiths,* and Jan Sjo 1 vall

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

It is well-established that electrochemical reactions can occur within the capillary of an electrospray (ES) devise coupled to a mass spectrometer. In fact, such reactions must occur to maintain charge balance during the ES process. However, electrochemical reactions occurring distal to the capillary as a result of the high potential applied to the capillary have not been thoroughly investigated. In the present communication, we show that electrochemical processes can occur on a high performance liquid chromatography (HPLC) column coupled to an ES capillary. On-column solvent electrolysis is proposed to generate free radicals, which can subsequently initiate analyte oxidation. Oxidation of steroid sulfates possessing a reactive double bond between C-5 and C-6 is demonstrated. The possibility of similar reactions occurring within peptides possessing a site of unsaturation is also considered. Electrospray (ES) mass spectrometry (MS) is now widely used by the biochemistry community.1 Capillary column high performance liquid chromatography (HPLC) interfaced directly to ESMS2,3 is becoming ever more important in proteomic and metabolomic applications.4 In the ES process, the ES capillary is raised to a high potential, usually 2-6 kV, and this brings about the possibility of electrochemical processes occurring within the ES capillary. In fact, if the ES interface is considered as a controlled current electrolytic flow cell, electrochemical processes must occur within the ES capillary to maintain charge balance during the ES process.5,6 Usually, for protein and peptide analysis, the ES capillary is raised to a high positive potential, and oxidation of solvent or analyte can occur at the capillary wall. For example,

2Cl-(aq) f Cl2(g) + 2e-

(1)

4OH-(aq) f O2(g) + 2H2O(l) + 4e-

(2)

* Author for correspondence. Phone: +46 8 728 7721. Fax: +46 8 339004. E-mail: [email protected]. (1) Griffiths, W. J.; Jonsson, A. J.; Liu, S.; Rai, D. K.; Wang, Y. Biochem. J. 2001, 355, 545-561. (2) Andren, P. E.; Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 867-869. (3) Gelpı´, E. J. Mass Spectrom. 2002, 37, 241-253. (4) Yates, J. R. J. Mass Spectrom. 1998, 33, 1-9. (5) Van Berkel, G. J.; Zhou, F. Anal. Chem. 1995, 67, 3643-3649. (6) Van Berkel, G. J. In Electrospray Ionization Mass spectrometry: Fundamentals, Instrumentation and Applications; Cole, R. B., Ed.; John Wiley and Sons: New York, 1997; pp 65-106.

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2H2O(l) f O2(g) + 4H+(aq) + 4e-

(3)

M(capillary metal) f Mn+(aq) + ne-

(4)

When the interface is operated in the negative ion mode, as is often the case for nucleotide or metabolite analysis, reduction will proceed at the capillary surface.7 For example,

Mn+(aq) + e- f M(n-1)+(aq)

(5)

CH3CO2H(aq) + 2H+(aq) + 2e- f CH3CHO(aq) + H2O(l) (6) Usually when discussing the positive ion ES process, the ES capillary is regarded as the anode (positive potential) and the “downstream” ES interface counter electrode as the cathode (negative potential).8,9 These electrodes are, of course, reversed when ES is operated in the negative ion mode. A potential difference will also be developed “upstream” between the ES capillary and a sample injection loop, syringe pump, or on-line HPLC column. Electrochemistry can also occur between these two “electrodes”, although this possibility is rarely considered.10,11 For example, when the ES interface is operated in the negative ion mode, solvent could be oxidized at the “upstream” anode, leading to the generation of molecular oxygen (3) or free radicals (7). For example,

H2O(l) f OH•(aq) + H+(aq) + e-

(7)

The production of free radicals can initiate a cascade of oxidation reactions. However, the oxidation reactions that occur at the anode will depend on the nature of the electrode (whether it can be readily oxidized itself), the species in solution available for oxidation, and the potential at the electrode. In most cases, “upstream” electrochemistry is not considered to effect the analyte being investigated and is ignored. In the course of a study on brain metabolites, we have coupled capillary column HPLC to a low-flow-rate ES interface (Figure 1a, (7) Van Berkel, G. J. J. Mass Spectrom. 2000, 35, 773-783. (8) Blades, A. T.; Ikonomou, M. G.; Kebarle, P. Anal. Chem. 1991, 63, 21092114. (9) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1991, 63, 19891998 (10) Konermann, L.; Silvia E. A.; Sogbein, O. F. Anal. Chem. 2001, 73, 48364844. (11) Liu, S.; Griffiths, W. J.; Sjo ¨vall, J. Procedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, June 2-6, 2002. 10.1021/ac026066p CCC: $25.00

© 2003 American Chemical Society Published on Web 01/07/2003

Figure 1. Capillary column HPLC interfaced to ESMS. The HPLC pump, injector, splitters A and B, and unions 1 and 2 were made of stainless steel. The pre- and analytical columns consist of a fused-silica capillary packed with C18 particles. The ES capillary was made of a gold-coated tapered fused-silica capillary. High voltage was applied to union 1. In the absence of additional grounding, the pre- and analytical columns were at potentials above earth.

arrangement A). Coupling of the analytical column to the ES capillary was via a stainless steel “zero-dead-volume” union (union 1). The high voltage, necessary for ES, was also applied to this union to give a “liquid junction”. The ES interface was operated in the negative ion mode, and a potential difference was generated between the two ends of the analytical column and also the two ends of the precolumn. The analytical and precolumns were packed in-house and periodically tested for chromatographic performance by injection of a steroid sulfate mixture containing dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) sulfate. The capillary column HPLC system was used in the analysis of steroid sulfates isolated from brain tissue and plasma. The injected steroid sulfates, synthetic as well as from biological samples, were detected as [M - H]- ion peaks. Initially, there was no indication of chemical degradation of the compounds. Remarkably, after ∼1 month of normal performance, the capillary column system appeared to deteriorate in that the [M - H]- ion peak for DHEA sulfate was no longer detected when the steroid sulfate standard mixture was injected. It was replaced by a series of peaks apparently corresponding to steroid oxidation products. Equally surprisingly, when the column system was isolated from the liquid junction by an earthed union (union 2 in Figure 1b, arrangement B), the “normal” expected spectrum showing the [M - H]- ion for DHEA sulfate was obtained. In this Technical

Note, we attempt to explain these findings and also to illustrate the potential value and hazard of “upstream” electrochemistry that can accompany the ES process. EXPERIMENTAL SECTION Chemicals. Steroid sulfates were from previous studies.12 Peptides were from Sigma (St. Louis, MO). Methanol and acetonitrile were from Rathburn Chemicals Ltd. (Walkerburn, U.K.). Ammonium acetate, D2O, and CD3OD were from Aldrich (Milwaukee, WI). H218O was from Larodan AB (Malmo¨, Sweden). Preparation of Capillary Columns. A packing procedure similar to that described by Alborn and Stenhagen13 was used. A small amount of coarse packing material (Bondesil, 40 µm, Varian, Walnut Creek, CA) was transferred to a piece of fused-silica capillary tubing (100 µm i.d., 375 µm o.d.), the end of which had been shrunk to 10-20-µm i.d. using a flame. This packing formed a 3-5 mm support on which the column was packed using a slurry (10 mg/mL) of C18 particles (Genesis 3 µm, Jones Chromatography, Mid Glamorgan, U.K.) in chloroform. Methanol was used as the pumping medium, and the pressure was increased to 400 bar in 1 min with a pneumatic pump (Maximator, Schmidt, Krantz (12) Griffiths, W. J.; Liu, S.; Yang, Y.; Purdy, R.; Sjo¨vall, J. Rapid Commun. Mass Spectrom. 1999, 13, 1595-1610. (13) Alborn, H.; Stenhagen, G. J. Chromatogr. 1985, 323, 47-66.

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& Co, Zorge, Germany). Upon completion of the packing, the methanol was replaced by water, which was pumped through the column overnight to compress the packing. The column was finally inspected under a microscope to check the homogeneity of the packing. Then the inlet and outlet of the column were sintered using a sinter device (InnovaTech Ltd, Stevenage, U.K.). Analytical columns were made with a length of ∼400 mm and precolumns with a length of ∼50 mm. Chromatography System. Shown in Figure 1 are schematic drawings of the chromatography system used in the present study. The basic system (Figure 1a, arrangement A) consists of two syringe pumps (ISCO model 100 DM, Lincoln, NE), a Valco C6 injector (Valco, Houston, TX), a Valco T (ZT1C, splitter A), a precolumn, a second Valco T (ZT1C, splitter B), the analytical column, and a Valco zero-dead-volume union (ZU1XC, union 1). The analytical column and union 1 could be mounted in the ES probe of an AutoSpec mass spectrometer. The injector, splitters, and zero-dead-volume union were made of stainless steel. The mobile phases in the pumps A and B were 10% and 90% aqueous methanol, respectively, both containing 10 mM ammonium acetate. Samples of 1-20 µL were injected using the Valco C6 injector with an external 20-µL loop. Electrospray Mass Spectrometry. Negative ion ES mass spectra were recorded on an AutoSpec-OATOFFPD doublefocusing instrument (Micromass, Manchester, England). Goldcoated tapered fused-silica capillaries of 8- or 15-µm tip size were connected to the zero-dead-volume union (union 1) and used as micro-ES capillaries (New Objective Inc., Woburn, MA). Electrical contact was made via this zero-dead-volume union (union 1) in a liquid junction to give a potential of approximately -5.5 kV on the ES capillary. The analytical column could be positioned within (Figure 1a, arrangement A) or external to the AutoSpec ES probe (Figure 1b, arrangement B). In the latter case, the analytical column was connected via a second zero-dead-volume union (union 2) and transfer tubing to union 1 to which the high potential was applied. Union 2 could be earthed. Negative ion ES mass spectra were also recorded on a Quattro Ultima triple quadrupole instrument (Micromass, Manchester, England), in which case the column arrangement was similar to that depicted in Figure 1b. The potential on the ES capillary applied via union 1 was approximately -2.5 kV. When using the AutoSpec instrument, the capillary, cone, and ring voltages were optimized for steroid sulfates and had values of approximately -5.5, -4.5, and -4.0 kV, respectively. The accelerating voltage was -4 kV. Low-resolution (3000, 10% valley definition) negative ion mass spectra were recorded by magnet scans over the m/z range of 340-600 at a rate of 10 s/decade. High-resolution (8000, 10% valley definition) spectra were recorded by voltage scans over a minimum m/z range at a rate of 10 s/scan. Tandem mass (MS/MS) spectra were recorded on the orthogonal acceleration (OA) time-of-flight (TOF) analyzer. The collision energy was 400 eV. Xenon was used as the collision gas at a pressure sufficient to attenuate the precursor ion beam by 75%. For experiments on the Quattro Ultima, the typical capillary and cone voltages were -2.5 kV and -90 V, respectively. A cone gas flow of 30 L/h was used. Negative ion mass spectra were recorded over an m/z range of 300-700 at unit mass resolution. 1024

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Routine HPLC Procedure. For the analysis of steroid sulfates from biological samples, the HPLC system was operated in the following way: 20 µL of sample was injected in a solution of 10% aqueous methanol with solvent A being pumped through the columns at 200 bar. Splitter A was closed and splitter B, open. After waiting the necessary period of time for the analyte to be sorbed on the precolumn, splitter B was closed, and splitter A was opened. Elution from the precolumn was initiated by starting either a gradient program in the flow control mode or the B pump in the pressure control mode. The sample was transferred to the analytical column, separated on the analytical column, and analyzed by ES mass spectrometry. It is necessary to regularly test the performance of capillary HPLC columns. A mixture of the sulfates of the following five steroids was used for this purpose: DHEA, pregnenolone (3βhydroxy-5-pregnen-20-one, PREG), 7-oxo-pregnenolone (3β-hydroxy-5-pregnene-7,20-dione, 7-oxo-PREG), epiallopregnanolone (3β-hydroxy-5R-pregnan-20-one), and allopregnanolone (3R-hydroxy-5R-pregnan-20-one). HPLC Procedure for the Characterization of Oxidation Products. For the characterization of oxidation products, the HPLC procedure was modified in that the HPLC system was run in isocratic mode, and all the splitters were closed. Samples were injected in 10, 50, or 90% aqueous methanol (or acetonitrile) and were eluted with 90% aqueous methanol (or acetonitrile); all solvents contained 10 mM ammonium acetate. For experiments to observe deuterium or 18O incorporation, sample injection and elution were made in labeled solvents. RESULTS Column Testing. It is essential to regularly test the performance of capillary HPLC columns that are used in metabolite analysis. Using the chromatography system depicted in Figure 1a (arrangement A), the mixture of five steroid sulfates was injected onto the column. A “good column” will allow separation of the five steroid sulfates with minimum peak tailing. To maximize analytical use of the mass spectrometer, tests can be performed on- or off-line. Tests were performed off-line by using a UV detector connected to union 1 in place of the ES capillary. In these tests, high voltage was not applied to union 1. Generally, a column performing well in the off-line test proves to be a “good column” in a subsequent ES performance test. Very surprisingly, a preand analytical column combination that had been used previously for metabolite analysis and performed satisfactorily failed a later ES test when incorporated into the chromatographic system in Figure 1a. The [M - H]- ions for DHEA sulfate, PREG sulfate, and 7-oxo-PREG sulfate were absent and were replaced by [Μ H]- ions of apparent oxidation products. The [M - H]- ions for the two saturated steroid sulfates remained unchanged. When the test mixture was independently analyzed by nano-ES from a metalcoated borosilicate capillary, the oxidation products were not observed, and the expected [M - H]- ions for each steroid sulfate were observed. When the off-line chromatography test was repeated (UV rather than ES detection), the peak shapes and the retention times of each steroid sulfate appeared normal. In combination, these results suggest that “upstream” on-column electrochemistry is occurring as a consequence of the high voltage connection at union 1 in arrangement A (Figure 1a). It appears

Scheme 1. Structures of Steroid Sulfates

that DHEA sulfate, PREG sulfate, and 7-oxo-PREG sulfate are undergoing on-column oxidation. Oxidation of Steroid Sulfates. An initial investigation of oncolumn oxidation was performed using DHEA sulfate, PREG sulfate, 7-oxo-PREG sulfate, allopregnanolone sulfate, cholesterol sulfate, and 17β-estradiol (estra-1,3,5(10)-triene-3,17β-diol) 3-sulfate as test compounds, each at a concentration of 20 ng/µL. DHEA sulfate, PREG sulfate, 7-oxo-PREG sulfate, and cholesterol sulfate possess a double bond in the B ring between carbons 5 and 6, while 17β-estradiol 3-sulfate has an aromatic A ring (Scheme 1). Samples were injected in 10, 50, or 90% aqueous methanol (or acetonitrile) and were eluted with 90% aqueous methanol (or acetonitrile); all solvents contained 10 mM ammonium acetate. Oxidation products were initially found when the HPLC system was connected to the AutoSpec mass spectrometer as in arrangement A (Figure 1a). When the system was changed to arrangement B (Figure 1b) with union 2 not earthed, the yield of oxidation products was reduced by 50%. With arrangement B connected to the Quattro Ultima instrument, the yield of oxidation products was reduced by 70%. When union 2 connecting the column outlet and the transfer capillary in arrangement B (Figure 1b) was earthed, oxidation products were not formed. In arrangement A (Figure 1a), grounding of the metal splitter B at the inlet of the analytical column prevented the formation of the oxidation products. Using the AutoSpec instrument and arrangement A (Figure 1.a), a voltage of -5.5 kV was applied to union 1, and a potential difference of 25 V was measured between the splitters A and B. This generated a current of 2.5 µA across the precolumn (107 ohm). When splitter B was grounded or, alternatively, the precolumn was short-circuited, the potential difference across the precolumn was eliminated, and the formation of oxidation products was prevented. When splitter A rather than splitter B was earthed, oxidation products were still formed. These results strongly suggest that the site of oxidation is the precolumn and that it is an electrochemically initiated process.

Figure 2. ES spectra of DHEA sulfate obtained with the HPLC arrangement A (Figure 1): (a) injection solvent 90% aqueous CH3OH, (b) injection solvent 90% aqueous CH3OH, (c) injection solvent 90% aqueous CD3OD, (d) injection solvent 10% aqueous CH3OH, and (e) injection solvent 10% CH3OH. Spectrum a was recorded with splitter B earthed; spectra b-e were recorded with no earth connection on the HPLC column system. Spectra a-d were recorded on the AutoSpec instrument. Spectrum e was recorded on the Quattro Ultima instrument.

Oxidation of DHEA Sulfate. Initial experiments were performed on the AutoSpec instrument. DHEA sulfate was the first steroid to be examined, because it is a major steroid in humans and has a relatively simple structure (Scheme 1). An ES spectrum of DHEA sulfate obtained with the HPLC arrangement A (Figure 1a) and with splitter B earthed is shown in Figure 2a. The spectrum is identical to that obtained by nano-ES from a metalcoated borosilicate capillary. When the earth connection to splitter B was removed, on-column oxidation of DHEA sulfate occurred. As is evident from Figure 2b-d, the abundance of different oxidation products varied with injection solvent. When the sample was injected in 10-50% aqueous methanol or in 50% aqueous CH3CN, the predominant peak was at 383 Th, that is, 367 + 16 (Figure 2d); however, when samples were injected in 90% aqueous methanol, peaks at m/z 397 (367 + 16 + 14) and 429 (367 + 62) became more significant (Figure 2b). When the sample was injected in 100% CH3CN, only very minor oxidation products were observed. These results indicate the participation of water and methanol in the reactions occurring under the different conditions. When injections were made in isotopically labeled solvents, the oxidation products shifted in mass. Injection in H218O/CH3CN (1:1, v:v) caused the oxidation products observed at m/z 383 with the unlabeled solvents to be shifted to m/z 385. This shows that 18O from H218O is incorporated into the oxidation product. Injection of DHEA sulfate in H2O/CD3OD or D2O/CD3OD (1:9, v:v) resulted in the ion previously observed at m/z 397 in the unlabeled solvents to be shifted to m/z 400 in the labeled solvent (Figure 2c). This suggests that CD3 from CD3OD is incorporated into this ion. Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

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Table 1. DHEA Sulfate Oxidation Productsa exptl m/z

chem formula

chem formula change

367.1579 381.1377c 383.1521 397.1683 399.1535d 401.1612 411.1838 413.1667 415.1791 427.1783 429.1947 441.1933 443.1830e 445.1887

C19H27O5S C19H25O6S C19H27O6S C20H29O6S C19H27O7S C19H29O7S C21H31O6S C20H29O7S C20H31O7S C21H31O7S C21H33O7S C22H33O7S C21H31O8S C21H33O8S

+O, -2H +O +O, +CH2 +2O +2O, +2H +O, +2CH2 +2O, +CH2 +2O, +2H, +CH2 +2O, +2CH2 +2O, +2H, +2CH2 +2O, +3CH2 +3O, +2CH2 +3O, +2H, +2CH2

proposed structure change

(i) saturated B ring and 6-oxo or 7-oxo, (ii) 5-6 epoxide 7-methoxy hydroperoxy saturated B ring diol saturated B ring hydroxy/methoxy bis methoxy saturated B ring bis methoxy

δ ppmb 0.0 0.0 2.7 1.2 14.4 2.2 0.8 8.2 0.1 1.8 2.8 8.8 20 2.1

a ESMS data for DHEA sulfate oxidation products was obtained with HPLC arrangement A (Figure 1a). Spectra were recorded on the AutoSpec. Difference in mass between experimental and calculated values. c Also present in synthetic DHEA sulfate. d Unresolved multiplet consisting of monoisotopic C19H27O7S and heavy isotopomers of C20H29O6S. e Unresolved multiplet consisting of monoisotopic C21H31O8S and heavy isotopomers of C22H33O7S.

b

Scheme 2. Fragmentation of DHEA Sulfate and Its Oxidation Products of m/z 383

Accurate mass measurements were made on the DHEA sulfate oxidation products to determine their elemental composition (Table 1). Experimental values were in agreement (10 ppm) with calculated values for proposed chemical formulas (Table 1). Exceptions were the ions at 399 Th and 443 Th. The peak at 399 was an incompletely resolved multiplet of C19H27O7S and heavy isotopomers of C20H29O6S. The peak at 433 was also an incompletely resolved multiplet consisting of C21H31O8S and heavy isotopomers of C22H33O7S. To identify the chemical structures associated with the molecular formula determined in Table 1, MS/MS experiments were performed on the AutoSpec-OATOF instrument. ES-MS/MS spectra of the precursor ions at m/z 383, 397, 413, 415, 427 and 429 were recorded. To record the MS/MS spectra of the ions of m/z 383, 413, and 415, 20 µL of DHEA sulfate in 50% aqueous acetonitrile or 50% aqueous methanol was injected. To record CID spectra of 397, 427, and 429, 20 µL of DHEA sulfate in 90% aqueous methanol was injected. A consequence of injection in “strong” chromatographic solvents was the loss of chromatographic performance. This was further degraded by isocratic elution in solvent B (90% aqueous methanol containing 10 mM ammonium acetate). Elution was performed in this solvent to minimize analyte retention on the column system and the possibility of further oxidation. The MS/MS spectra recorded on the AutoSpec-OATOF of the [M - H]- ion from unmodified DHEA sulfate at m/z 367 and the oxidation product 16 Th heavier at m/z 383 are shown in Figure 3a and b, respectively. Information from accurate mass measure1026

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ments indicates that the ion at m/z 383 differs from that at m/z 367 by the addition of an oxygen atom to the steroid skeleton (Table 1). No chromatographic separation of components of m/z 383 was achieved, and it is highly probable that the MS/MS spectrum shown in Figure 3b is a composite of more than one component (Scheme 2). The presence of fragment ions at m/z 191 ('b1) in the CID spectrum shown in Figure 3b indicates that the 5,6 double bond has been saturated in one of the components (compound 1).12,14 The neighboring peak at m/z 219 ('b2) suggests the presence of a second component (compound 2) incorporating an epoxide group between C-5 and C-6 (Scheme 2). The fragment ions at m/z 259 and 327 correspond to 'c1 and 'd1, respectively, for both compounds 1 and 2, whereas ions at m/z 303 and 285 are a result of the neutral loss of SO3 and H2SO4 from the precursors.12,14 The presence of the fragment ion at m/z 229 suggests the presence of a third component of unknown structure. When sample injection was made in CH3CN/H218O (1:1 v:v), the oxidation products incorporated 18O rather than 16O to give ions of m/z 385 rather than 383. In the MS/MS spectrum of m/z 385, the 'b1 fragment from compound 1 still appeared at m/z 191, but 'b2 from compound 2 now appeared at m/z 221 (Figure 3c). Ring fragment ions 'c1 and 'd1 were similarly shifted up by 2 Th in the spectra of the 18O-containing ion. These data suggest that the major oxidation products at m/z 383 (385 from H218O solvent) corresponds to a mixture of 3β-hydroxy-5ξ-androstane-7,17-dione 3-sulfate, that is, compound 1, and 3β-hydroxy-5,6ξ-epoxy-5ξ(14) Tomer, K. B.; Gross, M. L. Biomed. Environ. Mass Spectrom. 1988, 15, 89-98.

Figure 3. MS/MS spectra of (a) DHEA sulfate (m/z 367), injection solvent CH3OH:H2O (1:1); (b) DHEA sulfate oxidation product (m/z 383), injection solvent CH3OH:H2O (1:1); and (c) DHEA sulfate oxidation product (m/z 385), injection solvent CH3CN:H218O. The spectrum in a was recorded by nano-ES from a borosilicate needle; the spectra in b and c were micro-ES spectra using HPLC arrangement A (Figure 1a) and the AutoSpec-OATOF instrument. MS/MS spectra were recorded on the orthogonal acceleration (OA) time-of-flight (TOF) analyzer. The collision energy was 400 eV. Xenon was used as the collision gas at a pressure sufficient to attenuate the precursor ion beam by 75%.

androstane-17-one 3-sulfate, that is, compound 2. It is most probable that compounds 1 and 2 will each exist as two stereoisomers, one of which has the 5R and the other, the 5β configuration.15 In the absence of synthetic standard compounds, the identification of compounds 1 and 2 is not unequivocal. Alternative or additional structures for compound 1 are 3βhydroxy-5ξ-androstane-6,17-dione 3-sulfate. The CID spectrum and accurate mass measurements of the DHEA sulfate oxidation product(s) at m/z 397 indicate that the elements OCH2 have been added to the DHEA steroid skeleton in the form of a methyl ether group (Table 1). This is further supported by the CID spectrum of the equivalent ion (m/z 400) given by oxidation in aqueous CD3OD. CID spectra were also recorded of [M - H]- of m/z 415, 427, and 429. Using accurate mass and CID data together with information from heavy-isotopelabeling experiments, the structures given in Table 1 are suggested. Oxidation of Cholesterol Sulfate. When cholesterol sulfate ([M - H]- ) 465 Th) was injected on to the chromatography system (arrangement A, Figure 1a) in 70% aqueous methanol, major oxidation products were observed at m/z 481 (465 + 16) relative abundance (RA) 95%, 495 (465 + 30) RA 100%, 511 (465 + 46) RA 40%, 527 (465 + 62) RA 25%, and 543 (465 + 78) RA 25%. Cholesterol sulfate is considerably more hydrophobic than DHEA sulfate and was retained on the column system. Using a mobile phase of 90% aqueous methanol, some chromatographic separation of the cholesterol sulfate oxidation products was achieved. The ion at m/z 481 (465 + 16) is analogous to the ion m/z 383 (367 + 16) in the oxidation of DHEA sulfate. Shown in Figure 4 is the reconstructed ion chromatogram (RIC) for the ion at m/z 481. Four partially resolved peaks are observed, each of which was subjected to MS/MS, and the resulting spectra are compared to that of cholesterol sulfate (m/z 465), as shown in Figure 5. The MS/MS spectra of precursor ions of m/z 481 from peaks I and II are essentially similar and are shown in Figure 5b. The presence of the 'b1 ion at m/z 191 indicates saturation of the 5,6 double bond, whereas the ions at m/z 259 and 273 corresponding to 'c1 and 'c2 fragments, respectively, indicate that a ketone group is present at C-7 (or C-6) (Scheme 3). Further (15) Smith, L. L. Cholesterol Autoxidation; Plenum Press: New York, 1981.

fragment ions at m/z 327 ('d1), 353 ('e-15), 368 ('e), 396 ('f), 410 ('g), 424 ('h) and 438 ('i) are also consistent with a 7-oxodihydrocholesterol 3-sulfate structure. The two chromatographic peaks I and II are likely to represent the 5R and 5β isomers formed by saturation of the 5,6 double bond. The MS/MS spectra of chromatographic peaks III and IV are similar to one another but different from those of peaks I and II. In the MS/MS spectra of precursor ions of m/z 481 from peaks III and IV (Figure 5c), the 'b1 fragment ion is absent but replaced by a 'b2 ion at m/z 219. This indicates the presence of a 5,6-epoxide (Scheme 3). The remainder of the spectra shows a series fragment ions 'c1 (m/z 259)-'i (m/z 438) identical to those observed in the MS/MS spectra of m/z 481 from chromatographic peaks I and II, which are characteristic of B-ring oxidized cholesterol sulfate. These results suggest that the four oxidation product of m/z 481 correspond to 3β-hydroxy-5R-cholestan-7-one 3-sulfate and its 5β isomer (peaks I and II) and the isomeric 5,6R-epoxy-5β- and 5,6βepoxy-5R-cholestan-3β-ol sulfates. In the absence of reference standards, the above identification is not unequivocal; possible alternative structures for the compounds in peaks I and II are 3β-hydroxy-5ξ-cholestane-6,17-dione 3-sulfates. MS/MS spectra were also recorded on cholesterol sulfate oxidation products of m/z 495 and 527. The RIC for m/z 495 revealed two chromatographic peaks that gave similar MS/MS spectra. These spectra were interpreted to correspond to isomers of 7-methyl ethers of the 7R and 7β isomers of 5-cholestene-3β,7-

Figure 4. Cholesterol sulfate was injected into the HPLC system (arrangement A, Figure 1a) in 70% aqueous methanol and oxidation products were eluted with 90% aqueous methanol. The reconstructed ion chromatogram for m/z 481 is shown.

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Figure 5. MS/MS spectra of (a) cholesterol sulfate (m/z 465), (b) cholesterol sulfate oxidation product in HPLC peak I (m/z 481), and (c) cholesterol sulfate oxidation product in HPLC peak III (m/z 481). The spectrum in a was recorded by nano-ES from a borosilicate needle; the spectra in b and c were micro-ES spectra obtained using HPLC arrangement A (Figure 1a) with the AutoSpec instrument. The injection solvent was 70% aqueous methanol, and the eluting solvent was 90% aqueous methanol. MS/MS spectra were recorded on the OATOF analyzer. The collision energy was 400 eV. Xenon was used as the collision gas at a pressure sufficient to attenuate the precursor ion beam by 75%.

Scheme 3. Fragmentation of Cholesterol Sulfate and Its Oxidation Products of m/z 481a

a

The oxo group in the compounds of peaks I and II may be at C-6 or C-7.

diol 3-sulfates. The ion at m/z 527 was tentatively interpreted to a mixture of 5,6 and 5,7-dimethoxy-cholestan 3β-ol 3-sulfates. Autoxidation of steroids having the 3β-hydroxy-5-ene structure of cholesterol is a well-known process that has been reviewed in great detail by Smith.15,16 Following initial formation of radicals at different sites, a wide variety of products may be obtained. The major ones carry oxygen substituents at the allylic C-7, at C-5 and C-6, and in the side chain. The profiles of products differ, depending on the nature of the active oxygen species.16 In most cases, C-7 is thought to be the primary site of radical formation and oxygen attack. Oxidation of PREG Sulfate, 7-oxo-PREG Sulfate and 17βEstradiol 3-Sulfate. Like DHEA sulfate and cholesterol sulfate, PREG sulfate underwent a series of oxidation reactions when injected onto the column system (arrangement A). Oxidation products were observed in the ES spectra at m/z 411 (395 + 16) RA 50%, 425 (395 + 30) RA 100%, 441 (395 + 46) RA 10%, 443 (395 + 48) RA 20%, 455 (395 + 60) RA 10%, and 457 (395 + 62) (16) Smith, L. L. Chem. Phys. Lipids 1987, 44, 87-125.

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RA 50%. When 7-oxo-PREG sulfate was injected into the system, oxidation was again observed, but in this case, the peak corresponding to the unmodified 7-oxo-PREG-sulfate (m/z 409) was the base peak. This indicates that the 7-oxo-PREG sulfate is not as reactive as PREG sulfate, possibly because of the importance of C-7 as an initial site of oxidation.16 When 17β-estradiol 3-sulfate (Scheme 1) was injected, a very complex array of oxidation products was observed. The aromatic nature of 17β-estradiol 3-sulfate makes this steroid very susceptible to oxidation. Allopregnanolone Sulfate. Allopregnanolone sulfate (Scheme 1) contains a saturated steroid ring system. In the absence of carbon-carbon double bonds, oxidation products were not formed. Oxidation of Peptides: Tyr-Pro-Phe-Val-Glu-Pro-Ile/ Trp-His-Trp-Leu-Gln-Leu. Of the 20 commonly occurring amino acids, 4 are aromatic, that is, His, Trp, Tyr, and Phe. Considering that an aromatic steroid, that is, 17β-estradiol 3-sulfate, was highly susceptible to on-column oxidation, it seemed probable that peptides containing aromatic residues would be

Figure 6. ES spectra of peptides YPFVEPI and WHWLQL obtained with the HPLC arrangement A, injected in 70% aqueous methanol. Spectra were recorded on the AutoSpec instrument. (a) Negative ion spectrum of YPFVEPI, splitter B earthed. (b) Negative ion spectrum of YPFVEPI, no earth connection. (c) Positive ion spectrum of WHWLQL, splitter B earthed. (d) Positive ion spectrum of WHWLQL, no earth connection.

similarly sensitive to oxidation. To test this hypothesis, the peptides Tyr-Pro-Phe-Val-Glu-Pro-Ile (YPFVEPI) and Trp-His-TrpLeu-Gln-Leu (WHWLQL) were analyzed. When analyzed by nanoES from a gold-coated borosilicate needle or via the HPLC arrangement A with splitter B grounded, the major ions observed corresponded to the [M - H]- ion in the negative ion mode (Figure 6a) and the [M + H]+ ion in the positive ion mode (Figure 6c). When the earth connection to splitter B was removed and a potential difference was allowed to develop across the precolumn, the spectra changed markedly (Figure 6b and d) with the formation of a plethora of new ions. It is beyond the scope of the present study to identify these oxidation products; nevertheless, the spectra presented in Figure 6 clearly demonstrate the phenomena of on-column electrochemistry. Oxidation Observed Using the Quattro Ultima Mass Spectrometer. The potential applied to the ES capillary of the AutoSpec magnetic sector instrument when performing microES experiments is generally of the order 5-6 kV. In contrast, the capillary voltage necessary for similar micro-ES experiments on quadrupole based instruments is of the order of 2.5 kV. The fact that a lower potential was applied to union 1 (Figure 1) and that arrangement B was used in experiments using the Quattro triple quadrupole instrument resulted in a reduced potential difference between the ends of the analytical column and also across the precolumn. The consequence of this was that the formation of oxidation products when using the Quattro instrument was lower than when using the AutoSpec. However, the formation of oxidation products with HPLC arrangement B coupled to the Quattro is evident in Figure 2e. DISCUSSION The results of the current study demonstrate that electrochemical reactions in the column constitute a potential source of error in analyses performed by capillary HPLC/ESMS. In an unsatisfactory column system, unsaturated steroid sulfates can

undergo oxidation reactions when analysis is by negative ion ESMS. The nature and extent of these reactions depends on the analyte and the solvent used. Evidence from studies with isotopelabeled solvents indicates that electrolysis of water or methanol generates free radicals, which initiate oxidation. The extent of oxidation increases with increasing voltage applied to the ES capillary and decreases with increasing distance between the ES capillary and the site of the reactions. This indicates a dependence on a current through the site at which a reaction takes place. Experiments in which each union and splitter were systematically grounded show that the oxidation reactions occurred on the precolumn in the present system. This oxidation was prevented when a union or splitter between the precolumn and ES capillary was earthed. The precolumn and analytical column had previously been used in a study of plasma steroids in which sulfated steroids, initially isolated by solid-phase extraction with and without subsequent purification by anion exchange chromatography, were analyzed by capillary column HPLC using arrangement A. It seems likely that the precolumn has become activated by some material in the injected extracts. The nature of this material is not known. It is not clear whether column activation is specific for batches of column packing material or as a result of specific sample treatment. Whatever the reason for column activation, the fact that it can occur is evident from the results of the present study. With the increasing use of capillary column HPLC/ESMS and the desirability of connecting columns at the liquid junction or in the electrospray capillary itself,2,17,18 the possibility of on-column chemistry must be considered. It is advisable to test such column systems with compounds containing a reactive center of unsaturation. Since a column may be activated at any time, as illustrated in the present study, an HPLC/ESMS design that permits isolation of the column system from the ES capillary by an earth connection might be preferable in work with crude biological samples. When the nature of the column activation becomes elucidated, the column system in arrangement A might be used for the “combinatorial” synthesis of oxidation products. Several oxidized metabolites of DHEA, pregnenolone, and cholesterol are biologically active, some as ligands to nuclear receptors. Oxidation products could be collected by either spraying onto a metal plate or more simply by just collecting the eluates from the analytical column when zero voltage is applied to the ES capillary after first generating products on the column with the ES voltage on. In fact, Bruins and co-workers19,20 have used an on-line electrochemical flow cell in this manner for the collection or direct analysis of “upstream” electrochemically oxidized organic molecules, including peptides. The concept of “upstream” electrochemistry has recently been explored by Konermann et al.,10 who noted marked effects on protein folding as a consequence of pH changes within the ES capillary. Konermann et al.10 performed their study with the ES (17) Peng, J.; Gygi, S. P. J. Mass Spectrom. 2001, 36, 1083-1091. (18) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (19) Permentier, H.; Jurva, U.; Barroso, B.; Bischoff, R.; Bruins, A. 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando FL, June 2-6, 2002. (20) Jurva, U.; Wikstro ¨m, H. V.; Bruins, A. P. Rapid Commun. Mass Spectrom. 2002, 16, 1934-1940.

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interface in the positive ion mode and measured an “upstream potential” difference between the ES capillary and the earthed metal needle of the syringe used for sample injection. This resulted in electrochemical reduction of solvent at the metal needle of the syringe and, consequently, enhanced charge-balancing oxidation of water and lowering of pH at the ES capillary. This resulted in protein unfolding. The generation of an “upstream” potential difference in both Konermann’s study and the present work can thus have a marked effect on the appearance of ES mass spectra; however, appropriate grounding (or floating) of the instrumental setup can alleviate potential problems.

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ACKNOWLEDGMENT This work was supported by the Swedish Medical Research Council (grant no. O3X-12551), and Karolinska Institutet. The authors are grateful to Jon Watkins for assistance with voltage measurements. Access to the Quattro Ultima instrument was kindly provided by Micromass Nordic AB. Bjo¨rn A° kermark is thanked for helpful discussions. Received for review August 22, 2002. Accepted November 15, 2002. AC026066P