Exact Mass Measurements On-Line with High-Performance Liquid

Thomas Storm, Claudia Hartig, Thorsten Reemtsma*, and Martin Jekel. Department of Water Quality Control, Technical University of Berlin, Sekr. KF 4, S...
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Anal. Chem. 2001, 73, 589-595

Exact Mass Measurements On-Line with High-Performance Liquid Chromatography on a Quadrupole Mass Spectrometer Thomas Storm, Claudia Hartig, Thorsten Reemtsma,* and Martin Jekel

Department of Water Quality Control, Technical University of Berlin, Sekr. KF 4, Strasse des 17. Juni 135, 10623 Berlin, Germany

Exact mass measurements were performed on-line with high-performance liquid chromatography on a quadrupole mass spectrometer. Compounds with molecular weights from 98 to 797, mainly aromatic sulfonates and sulfonamides, were analyzed with electrospray ionization in positive or negative mode. Internal mass calibration compounds were continuously added after separation. A Gaussian fit of the mass errors of 808 individual measurements (concentrations of 1-10 mg/L, 20-200 ng absolute on column) resulted in a mean error of 0.1 mmu (0.45 ppm) and a standard deviation σ of 1.5 mmu (5.4 ppm). The 99.7% confidence intervals (3σ) were (4.5 mmu ((16.2 ppm) for single mass measurements. Averaging 10 measurements further reduced the errors to less than (1.5 mmu ((5 ppm). Isobaric interferences with ions resulting from the mass calibrants were avoided by the use of complementary mass calibrants. The results were verified (differences below (4.5 mmu) with a LC/ oa-TOFMS. Limited mass range chromatograms were used to enhance selectivity in the analysis of mixtures. The method was applied to determine the elemental composition of a potential dye metabolite detected in anaerobically treated textile wastewater. Exact mass measurements are employed on a routine basis for the confirmation of elemental compositions of organic compounds,1-5 and they are of great use in solving structure elucidation problems in synthetic organic or pharmaceutical chemistry.6,7 In environmental analysis, exact mass determinations have been successfully applied to the characterization of entirely * Corresponding author: (tel) +49 30 314 26429; (fax) +49 30 314 23850; (e-mail) [email protected]. (1) Perkins, G.; Pullen, F.; Thompson, C. J. Am. Soc. Mass Spectrom. 1998, 68, 546-551. (2) Haas, M. J. Rapid Commun. Mass Spectrom. 1999, 13, 381-383. (3) Huang, N.; Siegel, M. M.; Muenster, H.; Weissenberg, K. J. Am. Soc. Mass Spectrom. 1999, 10, 1212-1216. (4) Burton, R. D.; Matuszak, K. P.; Watson, C. H.; Eyler, J. R. J. Am. Soc. Mass Spectrom. 1999, 10, 1291-1297. (5) Huang, N.; Siegel, M. M.; Kruppa, G. H.; Laukien, F. H. J. Am. Soc. Mass Spectrom. 1999, 10, 1166-1173. (6) Lee, M. L.; Klohr, S. E.; Kerns, E. H.; Volk, K. J.; Leet, J. E.; Schroeder, D. R.; Rosenberg, I. E. J. Mass Spectrom. 1996, 31, 1253-1260. (7) Wolff, J.-C.; Mont, S.; Haskins, N.; Bell, D. Rapid Commun. Mass Spectrom. 1999, 13, 1797-1802. 10.1021/ac0006728 CCC: $20.00 Published on Web 01/03/2001

© 2001 American Chemical Society

unknown pollutants8,9 by GC/MS and to the identification of target compounds10 by LC/MS. Double focusing sector field-1-3,11 or Fourier transform ion cyclotron resonance mass spectrometers4,5 are the preferred instrumentation for exact mass measurements, since they are capable of high mass resolution (exceeding M/∆M ) 10.000), which helps to avoid errors due to interference of unresolved isobaric ions present in the spectrum from impurities or the internal mass calibrants. Accordingly, high mass resolution is often regarded to be a prerequisite for exact mass measurements and especially for measurements within the error limit of (5 ppm that is generally used as a benchmark12 in structure proofs. However, exact masses can be measured with comparable precision and accuracy on instrumentation only capable of low mass resolution such as single focusing sector field,13 quadrupole,14-16 and time-of-flight (TOF)10,17-20 mass spectrometers. Unresolved ions are more likely to affect exact mass determinations under low-resolution conditions,21 but low-resolution mass analyzers are generally far less expensive, are easier to operate than high-resolution instruments, and are readily interfaced with separation techniques such as GC, HPLC, or CE. Consequently, low-resolution mass spectrometers that can measure exact masses, especially TOF or hybrid quadrupole/TOFMS, have gained increasing popularity in many fields such as bioanalytical,20 combinatorial,22 and environmental chemistry.10 (8) Grange, A. H.; Sovocool, G. W., Donnelly, J. R.; Genicola, F. A.; Gurka, D. F. Rapid Commun. Mass Spectrom. 1998, 12, 1161-1169. (9) Vetter, W.; Alder, L.; Palavinskas, R. Rapid Commun. Mass Spectrom. 1999, 13, 2118-2124. (10) Hogenboom, R.; Niessen, W. M. A.; Little, D.; Brinkman, U. A. Th. Rapid Commun. Mass Spectrom. 1999, 13, 125-133. (11) McMurray, W. J.; Greene, B. N.; Lipsky, S. R. Anal. Chem. 1966, 38, 11941204. (12) Guidelines for authors. J. Am. Chem. Soc. 1999, 121, 7A-11A. (13) Hamar, C. G.; Hessling, R. Anal. Chem. 1971, 43, 298-306. (14) Roboz, J.; Holland, J. F.; McDowell, M. A.; Hillmer, M. J. Rapid Commun. Mass Spectrom. 1988, 2, 64-66. (15) Tyler, A. N.; Clayton, E.; Green, B. N. Anal. Chem. 1996, 68, 3561-3569. (16) Kostiainen, R.; Tuominen, J.; Luukkanen, L.; Taskinen, J.; Green, B. N. Rapid Commun. Mass Spectrom. 1997, 11, 283-285. (17) Zubarev, R. A.; Håkansson, P.; Sundqvist, B. Rapid Commun. Mass Spectrom. 1996, 10, 1386-1392. (18) Russel, D. H.; Edmondson, R. D. J. Mass Spectrom. 1997, 32, 263-276. (19) Cotter, R. C. Anal. Chem. 1999, 71, 445A-451A. (20) Palmer, M. E.; Clench, M. R.; Tetler, L. W.; Little, D. R. Rapid Commun. Mass Spectrom. 1999, 13, 256-263. (21) Blom, K. F. J. Am. Soc. Mass Spectrom. 1998, 9, 789-798. (22) Lane, S. J.; Pipe, A. Rapid Commun. Mass Spectrom. 1999, 13, 798-814.

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Despite the fact that quadrupole mass analyzers are among the most prevalent instruments for LC/MS or LC/MS/MS, there is no report on exact mass measurement on-line with liquid chromatography on such an instrument. At least, exact mass measurements with a quadrupole MS coupled to gas chromatography have recently been reported.23 In this paper, we describe exact mass measurements performed on-line with HPLC separations on a triple-quadrupole MS. The performance of the method in terms of mass measurement accuracy and sensitivity is described and critical factors are outlined. The results obtained for unknown compounds were verified independently with an ESIorthogonal acceleration (oa)-TOFMS. The method proved to be suitable for the determination of elemental compositions of unknown analytes present in treated textile wastewater or formed during biodegradation experiments. EXPERIMENTAL SECTION Chemicals and Reagents. The reactive dyes Reactive Black 5 (RB5) and Reactive Red 198 (RR198) were gifts from Dystar (Frankfurt, Germany), and Reactive Orange (RO16) was obtained from Aldrich (Deisenhofen, Germany). After hydrolysis,24 RB5 and RR198 were further purified by preparative HPLC, while the RO16 hydrolysate was used directly. Methylenebis-8,8′-naphthalene-2sulfonate was provided by C. Wolf (DVGW-TZW, Karlsruhe, Germany). 2-Aminonaphthalene-1-sulfonate and naphthalene-1,5disulfonate were donated by the Bayer AG (Leverkusen, Germany). All other standards were commercially available and used as received. Sodiated lauryl ether sulfate (trade name Genapol LRO) was obtained from Hoechst (Darmstadt, Germany). Alkanesulfonates, perfluoroctanesulfonate, and poly(ethylene glycol) n-alkyl-3-sulfopropyl diethers were available from Aldrich. Reagents and HPLC eluents were of reagent grade or better. Textile wastewater was supplied by a dye house in Berlin (Germany), and sulfonamide metabolites were detected in freeze-dried and reconstituted samples from biodegradation experiments. HPLC Conditions. Separations were performed on HP1100 HPLC systems (Hewlett-Packard, Waldbronn, Germany). For analytes detected in positive ion mode, a binary gradient on a 50 × 2 mm column filled with 3-µm Luna C18(2) (Phenomenex, Aschaffenburg, Germany) was used. The solvent system consisted of 3% acetonitrile as eluent A and 75% acetonitrile as eluent B, both acidified with 1% formic acid. A linear gradient from eluent A to eluent B in 8 min with a flow rate of 0.25 mL/min was employed. After an isocratic hold for 1 min, the system was returned from eluent B to eluent A in 0.5 min and equilibrated for 5.5 min. The analytes detected in negative ion mode were separated on a 150 × 2 mm column with 3-µm Luna phenylhexyl material. Here, eluent A was 30% methanol and eluent B was 70% methanol, both containing 1 mmol of acetic acid with 1 mmol of tributylamine as volatile ion-pairing agent.25 The gradient conditions were as follows: linear rise from 0 to 50% B in 20 min, to 100% B in 1 min, hold for 2 min, back to 0% B in 1 min, and equilibration for 6 min. The flow rate was 0.2 mL/min. (23) Fiehn, O.; Kopka, J.; Trethewey, R. N.; Willmitzer, L. Anal. Chem. 2000, 72, 3673-3580. (24) Karcher, S.; Moscato, I.; Hofmann, C.; Jekel, M. Vom Wasser 1999, 93, 265-278. (25) Storm, T.; Reemtsma, T.; Jekel, M. J. Chromatogr., A 1999, 854, 175-185.

590 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001

Figure 1. Comparison of resolutions (M/∆M) measured on the quadrupole MS and the oa-TOFMS for calibrant ions. Results for ESI+ in open symbols and for ESI- in filled symbols. Quadrupole MS: (O) m/z 125-325; (4) m/z 300-800; (b) m/z 135-335; (2) m/z 375-535; (() m/z 675-850. oa-TOFMS: (3) m/z 50-650; (1) m/z 100-1000.

A model 64 isocratic pump (Knauer, Berlin, Germany) with a model LP-21 pulse damper (Supelco, Deisenhofen, Germany) was used for postcolumn addition of mass calibrants to the quadrupole MS. The calibrants were added either via a 50-µL static mixer (Supelco) or a PEEK T-piece at a flow rate of 0.1 mL/min. For exact mass measurements on the oa-TOFMS, the internal lock mass was introduced with a model PHD 2000 syringe pump (Harvard, Holliston, MA). Mass Spectrometry. The quadrupole MS used in this study was a Quattro LC (Micromass, Altrincham, U.K.) with Z-spray source. Electrospray ionization (ESI) was used in positive and negative ion modes. As high digitization rates are essential to obtain exact peak centers,15 data were collected with 128 data points per m/z unit. Scan ranges of 180-230 mass units were employed. These were scanned in 5 s in continuum mode. The range itself depended on the mass of the analytes and was chosen to allow “bracketed” mass calibration of the analytes. Each scan range had to be tuned individually for optimal resolution and sensitivity. The resulting instrument specific tuning parameters were low-mass resolution 25, high-mass resolution 14.5, and ion energy 0.8 for low molecular weight analytes and low-mass resolution 24, high-mass resolution 13, and ion energy 0.6 for compounds of medium molecular weight. These parameters resulted in increasing mass resolutions with increasing mass ranging from 200 to 400 (10% valley definition) at the lower end of the mass range up to 1800 at the upper end of the mass range (Figure 1). For positive ionization, poly(ethylene glycol)s (PEGs), poly(ethylene glycol) monomethyl ethers (PEGMMEs), and poly(propylene glycol)s (PPGs) of various average molecular weights were used as internal mass calibrants. Solutions of 0.5-1 mg/L

Figure 2. Error distribution for 808 exact mass measurements on-line with HPLC in terms of (a) mmu and (b) ppm. Analyte mass range of m/z 157-796, measurements in positive and negative ESI modes.

were prepared in 1:1 water/methanol for postcolumn addition. For negative ionization, sodiated lauryl ether sulfate (SLES), poly(ethylene glycol) n-alkyl-3-sulfopropyl diethers (PEGSPEs) and, since these polymers do not give abundant ions below m/z 265, C4-C18 n-alkanesulfonates were used as internal mass calibrants. Concentrations for the polymers were 0.5-1 mg/L, respectively, and 0.01 mg/L for the n-alkanesulfonates in 1:1 water/methanol. Mass calibration of single analytes was performed by averaging at least eight scans over the chromatographic peak, with subsequent background subtraction, peak centering, and mass calibration of the spectrum, including a minimum of four reference peaks “bracketing” the analyte peak. To calibrate entire chromatographic runs, the “all file accurate mass” option of the MassLynx 3.3 software package (Micromass) was applied. LC/oa-TOFMS was performed on a LCT (Micromass) oaTOFMS with a Z-spray source. As on the Quattro LC, analyses were performed in positive and negative ion ESI modes. The oaTOFMS was calibrated with suitable mass reference compounds (a PEG mixture in positive ion mode or a mixture of alkanesulfonates, SLES, and PEGSPE in negative ion mode) and mass resolutions from 1900 to 3700 (10% valley definition) were obtained in the mass range from m/z 89-893 (Figure 1). For exact mass measurements a reference compound was added postcolumn and used as lock mass. With the LCT mass spectrometer, the signal intensity of the lock mass has to remain within certain limits (between 100 and 500 counts/scan) to prevent detector saturation phenomena. It has been recognized that the ascending amount of organic modifier in the mobile phase during gradient elution can drastically amplify the signal intensity of the lock mass.22 Leucine-enkephalin as a lock-mass produced too high intensities during the gradient conditions chosen for negative ion detection and was replaced by perfluoroctanesulfonate. For positive ions, tributylamine was used as a lock mass. RESULTS AND DISCUSSION Mass Measurement Accuracy and Error Limits. The number of possible elemental compositions that corresponds to

a measured mass increases with the molecular mass and with the uncertainty in the mass measurement.26 To reduce this number, low error limits are mandatory and the error limit should be reported along with exact mass measurement results. An error limit of (5 ppm is widely accepted for the confirmation of elemental compositions by mass spectrometry.12 However, to obtain unique elemental compositions for structure elucidation, wider or stricter limits may be required, depending on the molecular weight of the analyte and further knowledge about its composition.27 To assess the error limits of the exact mass measurement with the quadrupole instrument coupled to HPLC, the mass errors observed in 808 individual measurements of analytes with known elemental compositions were compiled. The measurements included analytes in the mass range from m/z 157 to 796, with the ionization performed in both positive and negative modes. In Figure 2, the error distribution is presented in terms of mmu and ppm. A Gaussian fit results in a mean error of 0.1 mmu (0.45 ppm), indicating a good mass measurement accuracy and the absence of grave systematic errors. The distribution of relative mass errors (ppm, Figure 2b) appears to be less homogeneous, possibly a result of the uneven distribution of the measured masses over the whole mass range (m/z 160-800). With a standard deviation (σ) of (1.5 mmu ((5.4 ppm), a 99.7% confidence interval (3σ) of (4.5 mmu ((16.2 ppm) can be established as a suitable measure of the precision of the method.28 These error limits are higher than those previously reported in an exact mass measurement study on a comparable quadrupole MS (1.1 mmu, 4.5 ppm).15 However, compared to that study, in which pure analytes were introduced into the MS by infusion, lower analyte concentrations, larger scan ranges, and a different data acquisition mode (continuum mode in contrast to multichannel acquisition) were (26) Biemann, K. In Methods in Enzymology; McCloskey, J. A. Ed.; Academic Press: San Diego, CA, 1990; Vol. 193, pp 295-305. (27) Gross, M. L. J. Am. Soc. Mass Spectrom. 1994, 5, 57. (28) Sack, T. M.; Lapp, R. L.; Gross, M. L. Int. J. Mass Spectrom. Ion Processes 1984, 61, 191-213.

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Table 1. Exact Mass Measurements On-Line with HPLC of 12 Test Compounds in Positive Ion Electrospraya m/z no.b

composition (M + H)+

calcd

mean (n ) 10)

mean error

mmu (ppm) (SD

max error

1 2 3 4 5 6 7 8 9 10 11 12

C10H11N4O2S C9H10N3O2S2c C11H13N4O2S C12H15N4O2S C10H13N3O3S C14H13N4O2S C12H15N4O4S C15H15N4O2S C18H15N4O5S C29H37O10d C31H39O11d C37H68NO13

251.0603 256.0214 265.0759 279.0916 254.0599 301.0759 311.0815 315.0916 399.0763 545.2386 587.2492 734.4690

251.0605 256.0225 265.0758 279.0907 254.0603 301.0772 311.0808 315.0915 399.0767 545.2400 587.2493 734.4679

0.2 (0.9) 1.1 (4.3) -0.1 (-0.3) -0.9 (-3.1) 0.4 (1.4) 1.3 (4.2) -0.6 (-1.9) -0.1 (-0.4) 0.4 (1.0) 1.4 (2.7) 0.1 (0.2) -1.1 (-1.7)

1.0 (4.0) 1.0 (3.6) 1.6 (6.1) 1.2 (4.3) 2.2 (8.7) 2.8 (9.5) 1.3 (4.2) 2.5 (7.8) 1.6 (4.1) 1.6 (2.9) 1.1 (1.9) 2.5 (3.4)

-1.6 (-6.3) 3.0 (11.7) 3.0 (11.3) -2.7 (-9.7) 4.9 (19.3) 6.6 (21.9) -3.3 (-10.8) -4.3 (-13.7) 2.2 (5.5) 3.5 (6.4) 1.9 (3.3) -4.0 (-5.4)

a Aliquots of 20 µL each of 1 mg/L solutions (20 ng absolute) were injected, using PEG as the mass calibrant. b Key: 1, sulfadiazine; 2, sulfathiazole; 3, sulfamerazine; 4, sulfamethazine; 5, sulfamethoxazole; 6, sulfadoxine; 7, sulfaquinoxaline; 8, sulfaphenazole; 9, sulfasalazine; 10, 10-desacetylbaccatine III; 11, baccatine III; 12, erythromycine. cA 20-µL aliquot of 10 mg/L solutions injected (200 ng absolute on column). d A 20-µL aliquot of 5 mg/L solutions injected (100 ng absolute on column); PPG as calibrant.

Table 2. Exact Mass Measurements On-Line with HPLC of 18 Test Compounds in Negative Ion Electrospraya m/z no.b

composition (M - H)-

calcd

mean (n ) 10)

mean error

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

C6H6NO3S C10H8NO3S C10H8NO3S C6H5O3S C10H7O6S2c C10H7O6S2c C10H7O4S C7H7O4S C6H4NO4S C10H8NO3S C10H7O3S C10H7O3S C10H7O4S C14H7O5S C21H13O6S2 C20H18N3O8S2 C26H24N5O13S4c C27H22N7O14S4c

172.0068 222.0225 222.0225 156.9959 286.9684 286.9684 223.0065 171.0116 201.9810 222.0225 207.0116 207.0116 223.0065 287.0014 427.0310 492.0530 742.0253 796.0108

172.0068 222.0224 222.0220 156.9964 286.9690 286.9682 223.0063 171.0123 201.9803 222.0220 207.0109 207.0110 223.0064 287.0019 427.0309 492.0527 742.0260 796.0114

-0.0 (-0.1) -0.1 (-0.5) -0.5 (-2.2) 0.5 (2.9) 0.6 (2.0) -0.2 (-0.6) -0.2 (-0.9) 0.7 (4.0) -0.7 (-3.7) -0.5 (-2.0) -0.7 (-3.4) -0.6 (-3.2) -0.1 (-0.5) 0.5 (1.7) -0.1 (-0.3) -0.3 (-0.6) 0.7 (0.9) 0.6 (0.7)

mmu (ppm) (SD 1.5 (9.1) 1.6 (7.0) 1.2 (5.8) 1.6 (5.8) 1.1 (3.9) 1.3 (4.4) 1.3 (7.0) 1.3 (7.6) 0.9 (4.5) 1.1 (5.1) 1.1 (5.1) 1.3 (6.1) 1.0 (4.5) 1.3 (4.7) 2.7 (5.0) 1.3 (2.7) 2.4 (3.7) 2.1 (2.8)

max error 4.1 (23.8) 2.4 (10.8) -2.4 (-10.8) 2.8 (17.8) 3.2 (11.2) 2.7 (9.4) -2.6 (-11.7) 2.4 (14.1) -1.6 (-7.9) -1.7 (-7.7) -2.4 (-11.6) -2.5 (-12.1) -2.3 (-10.3) 2.6 (9.1) -4.3 (-10.1) 2.0 (4.1) 5.5 (7.4) -3.5 (-4.4)

aAliquots of 20 µL each of 1 mg/L solutions (20 ng absolute) were injected. b Key: 1, 3-aminobenzenesulfonate; 2, 1-aminonaphthalene-7sulfonate; 3, 1-aminonaphthalene-5-sulfonate; 4, benzenesulfonate; 5, naphthalene-1,5-disulfonate; 6, naphthalene-2,6-disulfonate; 7, 1-hydroxynaphthalene-4-sulfonate; 8, toluene-4-sulfonate; 9, 3-nitrobenzenesulfonate; 10, 1-aminonaphthalene-8-sulfonate; 11, naphthalene-1-sulfonate; 12, naphthalene2-sulfonate; 13, 1-hydroxynaphthalene-2-sulfonate; 14, anthraquinone-2-sulfonate; 15, methylenebis-8,8′-naphthalene-2-sulfonate; 16, RO16; 17, RB5; 18, RR198. cA 20-µL aliquot of 5 mg/L solutions injected (100 ng absolute on column).

necessary when the measurements were performed on-line with HPLC. Nevertheless, the mass accuracy obtained here is slightly better than recently reported for exact mass measurement with quadrupole MS coupled to GC,23 where an average mass error of 5 mmu (25-8 ppm) and a standard deviation of 3 mmu (15-5 ppm) was achieved. For 30 test compounds, the results of 10 measurements were averaged and compared to the calculated masses. The majority of the test compounds were aromatic sulfonamides in positive ion mode and aromatic sulfonates in negative ion mode. Additional compounds were chosen to cover the mass range up to ∼800 Da. The results are summarized in Table 1 and Table 2 for measurements in positive and negative ion modes, respectively. Unless stated otherwise in Table 1 or 2, a concentration of 1 mg/L (20µL injections, 20 ng absolute) was suited to obtain mass measure592 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001

ment errors within the (5 ppm benchmark12 for exact mass determinations. The maximum error in 1 of 10 measurements typically ranged below 3 mmu and never exceeded 6.6 (Table 1, positive ion mode) and 5.5 mmu (Table 2, negative ion mode). Thus, even single measurements may be sufficiently exact in some cases. Isobaric Interferences. However, larger mass errors were observed under certain circumstances, and modifications were necessary for some compounds to meet the (5 ppm criterion. One source of these larger errors were isobaric interferences from ions introduced by the mass calibrants. For example, in the exact mass measurement of sulfathiazole (compound 2 in Table 1, m/z 256.0214) large errors (+7.8 mmu (+30.5 ppm) at 1 mg/L, and +1.3 mmu (+5.2 ppm) at 5 mg/L) were caused by an ion at m/z 256.1079 when PEG was used as calibrant. Only with a concentra-

Figure 3. Interference of the mass calibrant PEG analyzed at 25 (b) and 55 V (c) with the (M + H)+ of the analyte baccatine III (a) at m/z 587.25 (dotted line). With PEGMME (d) and PPG (e), no isobaric interference occurs.

tion of 10 mg/L did the error of the mean of 10 measurements remain below the (5 ppm margin (Table 1). Figure 3 illustrates the use of complementary calibrants to avoid such isobaric interferences. When PEG was used as the mass calibration substance in the exact mass determination of baccatine III, a large mass error of over 10 mmu was observed. This error resulted from an isobaric doubly charged ion at m/z 587.3 at low cone voltage (Figure 3b) or the (M + 2) isotope peak of a (M - H2O + H)+ fragment of PEG at m/z 587.4 at high cone voltage (Figure 3c). Neither PEGMME (Figure 3d) nor PPG (Figure 3e) interferes with the molecular ion of baccatine III (dotted line in Figure 3). Hence, exact mass determinations with PPG as the mass calibrant resulted in mass measurement errors below 5 ppm (compound 11, Table 1). In general, with the resolutions accessible for the quadrupole MS, mass calibrant ions that are isobaric with the analytes have to be avoided. As illustrated in Figure 3, this is possible for any analyte across the mass range by using polymers with different end groups or repeating units (such as PEG, PPG, and PEGMME),15 or homologous series of compounds such as the alkanesulfonates in negative ion mode, where interfering compounds can be deliberately excluded. SLES and PEGSPE provide ions at the same mass, but reference compounds have been described15,29,30 for negative ion ESI that can be used as a substitute for these calibrants. Influence of the Signal Intensity. The signal intensity of the analyte is another important factor that influences the quality of the exact mass measurement. The peak shape can deteriorate at lower intensities, thus leading to larger errors in the peak centering process. As mentioned before (see Tables 1 and 2), some substances require concentrations above 1 mg/L to achieve mass errors that meet the (5 ppm criterion. For example, for naphthalene-1,5-disulfonate, mass measurement errors below (5 (29) Cody, R. B.; Tamura, J.; Musselman, B. D. Anal. Chem. 1992, 64, 15611570. (30) Fujiwara, H.; Chott, R. C.; Nadeau, R. G. Rapid Commun. Mass Spectrom. 1997, 11, 1547-1553.

Figure 4. Differences (∆M) of the exact masses of unknown compounds determined with the LC/quadrupole-MS and LC/oaTOFMS: (A1-A3, B1-B2, C1-C3) molecular anions and fragments of compounds A-C (Figure 6); (D, E) unknown compound of nominal mass m/z 450 in textile wastewater; (F, G) metabolites of aromatic sulfonamides formed during degradation experiments (nominal masses of m/z 98 and 156).

ppm could only be achieved for concentrations of 10 and 5 mg/L (errors 3.8 and 2.1 ppm) but not for the 1 mg/L concentration (error 6.2 ppm). This was attributed to the generally lower response factors of disulfonates as compared to monosulfonates25 in electrospray ionization. The sensitivity in exact mass determinations is lower than in normal scanning mode because of the higher resolution required and because of the necessity to obtain excellent peak shapes. This loss of sensitivity may in part be offset by the enhanced selectivity obtained in limited mass range chromatograms, which should reduce the chemical noise. Nevertheless, concentrations of 1 mg/L were demonstrated to be sufficient in most cases yet investigated (Tables 1 and 2). Concentrations around 1 mg/L (or analyte amounts of ∼1 µg absolute) may be present in or may be obtained by extraction of a wide variety of environmental samples. Verification with LC/oa-TOFMS. In addition to repeated analyses of known compounds, the quality of exact mass determination by the LC/quadrupole MS was verified by parallel analysis of several unknown compounds by LC/oa-TOFMS (see Figure 4). LC/oa-TOFMS has recently emerged as a sensitive and reliable technique for exact mass measurements.31 It offers superior resolution (3-10-fold, Figure 1) and higher sensitivity in scanning mode10 compared to quadrupole MS. The exact masses of 12 compounds from LC/MS analyses of textile wastewater and biodegradation experiments were determined by both methods, and the mass difference never exceeds 4.5 mmu (Figure 4). This good agreement demonstrates the reliability of the exact mass measurements on the quadrupole MS. Finally, no isobaric interferences occurred on the quadrupole MS that were resolved on the oa-TOFMS. Applications. After calibrating all mass spectra in one chromatographic run, restricting the mass range to the 99.7% confidence interval of (4.5 mmu ((16.2 ppm) yields “limited mass range chromatograms”. These provide enhanced selectivity for the analysis of mixtures, as illustrated in Figure 5 with an HPLC/ MS chromatogram of a 1 mg/L standard mixture of aromatic sulfonates. The extracted ion current from m/z 222.8 to 223.8, (31) Eckers, C.; Wolff, J.-C.; Haskins, N. J.; Sage, A. B.; Giles, K.; Bateman, R. Anal. Chem. 2000, 72, 3683-3688.

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Figure 5. Chromatogram of a sulfonate standard mixture at 1 mg/L (20 ng absolute of each compound). The limited mass range chromatogram (a) of 223 ( 16.2 ppm provides higher selectivity than the usual unit mass resolution (b) of m/z 222.800-223.800. (c) RIC.

which would correspond to a typical extracted ion chromatogram under routine low-resolution conditions, displays five chromatographic peaks (Figure 5b). These stem from the molecular anions of two hydroxynaphthalenesulfonates (C10H7SO4, m/z 223.0065, peaks B and D, Figure 5), the (M + 1)sisotopic peaks of two aminonaphthalenesulfonates (the main contribution coming from 13CC9H8NSO3, m/z 223.0259, peaks A and C, Figure 5b) and the (M - SO2-H)fragment of anthraquinone-2-sulfonate (C14H7O3, m/z 223.0395, peak E, Figure 5b). The relative mass differences between the latter anions and the molecular anions of the hydroxynaphthalenesulfonates is 87 and 148 ppm, respectively. To resolve these mass differences by mass spectrometry alone, resolutions of 11 495 and 6758 (10% valley definition) would have been required, clearly exceeding the resolution obtainable on a quadrupole MS. However, chromatographic separation supplemented by high-mass accuracy under low-mass resolution conditions allows the selective detection of the hydroxynaphthalenesulfonates in the limited mass range chromatograms (Figure 5a). For the identification of unknowns by exact mass measurements, all elemental compositions have to be considered that fall within the error margins of the measurement.26,27 Depending on the molecular mass, the total number of elemental compositions may be large. To narrow down the number of possibilities, the minimal and maximal abundance of carbon and sulfur in the elemental composition can be deduced from the isotopic pattern. Furthermore, the exact masses of fragments at lower m/z values can be used to determine a unique elemental composition of the molecular ion. In Figure 6, the chromatogram of a sample of anaerobically treated textile wastewater is shown. The peaks A-C are unknown compounds formed during the anaerobic treatment. Compound A was not removed in a subsequent aerobic treatment and was thus of special interest in the following identification process. The mass spectrum of compound A (Figure 7 b) displays three peaks. Their exact masses were determined to be m/z 309.0190 ((1.2 mmu, n ) 5), 229.0602 ((1.7 mmu, n ) 5), and 187.0496 ((2.0 mmu, n ) 5). As shown in Figure 4 (data points A1-A3), these values were in good agreement with LC/oa-TOFMS measurements. 594 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001

Figure 6. Chromatogram of anaerobically treated textile wastewater (a) UV-absorption 225-235 nm; (b) RIC m/z 135-355; (c) extracted ion current m/z 308.8-309.8.

Figure 7. Mass spectra of peak A from Figure 6: (a) with mass calibrants (*); (b) after background subtraction (for exact masses refer to Table 3).

Since the (M + 1) and (M + 2) ions of the molecular ion at m/z 309 have relative intensities of 13 and 7%, respectively, the number of carbon atoms can be limited to 8-14 and the number of sulfur atoms to 1-2. With these limitations imposed, the 99.7% confidence interval of (4.5 mmu includes six possible elemental compositions (Table 3). The isotope pattern of the two fragments allows limiting the number of carbon atoms to 7-12 for fragment 1 and to 6-10 for fragment 2 (Table 3). This yields four possible elemental compositions for fragment 1 and one for fragment 2. Additionally, the exact mass differences between the molecular ion and fragment 1 and between the two fragments can be used to identify substructures of the compound; the lower masses of the differences make a unique elemental composition more likely. The mass differences of m/z 79.9587 and 42.0106 found here can only be due to SO3 (error 2.1 mmu, 25.7 ppm) and C2H2O (error 0.0 mmu, 0.5 ppm), respectively. These elemental compositions are unique within a limit of (9 mmu (2 times the 99.7% confidence interval).

Table 3. Determination of the Elemental Composition of an Unknown Constituent (Compound A in Figure 6) in Anaerobically Treated Textile Waste Water, Using Exact Masses and the Isotopic Patterna exact mass ((SD, n ) 5) 309.0190 (( 0.0012)

229.0602 (( 0.0017)

187.0496 (( 0.0020) 79.9587 (( 0.0018) 42.0106 (( 0.0030)

isotopic information

possible formulas

error mmu (ppm)

molecular anion (m/z 309) C, 8-14; C13H5N6O2S S, 1-2 C12H9N2O6S C10H9N6O2S C9H13N2O4S C9HN12S C8H5N8O2S2

-0.5 (-1.5) 1.0 (3.1) -3.8 (-12.4) -2.5 (-8.1) 2.2 (7.2) 3.6 (11.5)

fragment 1 (m/z 229) C, 7-12; C12H9N2O3 S, 0-1 C9H13N2O3S C8H5N8O C7H9N4O5

-1.1 (-4.8) -4.5 (-19.6) 1.6 (6.8) 2.9 (12.7)

fragment 2 (m/z 187) C, 6-10; C10H7N2O2 S, 0-1

-1.2 (-6.5)

molecular anion - fragment 1 SO3 fragment 1 - fragment 2 C2H2O

2.1 (25.7)

0.0 (0.5)

a The finally derived elemental compositions are printed in boldface type. (For mass spectrum, refer to Figure 7b.)

Finally, there is only one elemental composition for the molecular ion and for each of the fragments consistent with these results (printed in boldface type in Table 3). Thus, the combination of exact mass determination with the isotopic information present in the mass spectra allows the unambiguous identification of elemental compositions of entirely unknown low molecular weight compounds by LC/quadrupole-MS.

CONCLUSIONS Exact mass measurements can be performed on-line with HPLC on a quadrupole MS. The mass errors for single measurements is within a 99.7% confidence interval of (4.5 mmu ((16.2 ppm). By repetitive measurements (n ) 5-10) errors below (5 ppm can be obtained, thereby meeting widely accepted error limits for exact mass measurements. The limited mass resolution of 200-1800 (10% valley definition) requires careful selections of the mass calibration substances to avoid isobaric interferences, and a complementary set of mass calibrants is necessary to address the whole mass range. Concentrations of 1 mg/L (20 ng injected on column) proved to be sufficient in many cases. The method is, thus, well applicable for the confirmation of elemental compositions or structure elucidation in many areas of environmental analysis. This approach adds the option of exact mass determinations to the other mass and tandem-mass spectrometric detection schemes accessible on the triple-quadrupole MS to be used in combination with HPLC. ACKNOWLEDGMENT We are especially grateful to T. Gude and M. Schmitz (Schering AG, Berlin) for making available the LC/oa-TOFMS and to J. Spickermann (Micromass Europe) for technical advice. We thank Bayer AG (Leverkusen), C. Wolf (DVGW-TZW, Karlsruhe) and S. Karcher (TU Berlin) for providing reference compounds and M. Borchert (TU Berlin) for providing wastewater samples. The work was funded by the German Research Council (DFG, Bonn) through “SFB 193 ‘Biological treatment of industrial wastewater’ project A14”.

Received for review June 13, 2000. Accepted November 8, 2000. AC0006728

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