MS for Sensitive and

Downloaded by UNIV OF GUELPH LIBRARY on September 2, 2012 | http://pubs.acs.org Publication Date: May 20, 2003 | doi: 10.1021/bk-2003-0850.ch007

Chapter 7

HPLC/TOF-MS: An Alternative to LC/MS/MS for Sensitive and Selective Determination of Polar Organic Contaminants in the Aquatic Environment 1

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M . J. Benotti , P. Lee Ferguson , R. A. Rieger , C. R. Iden , C. E. Heine , and B. J. Brownawell 1

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Marine Sciences Research Center, Stony Brook University, Stony Brook, NY 11794-5000 Department of Pharmacology, Stony Brook University, Stony Brook, NY 11794-8651 Micromass, Inc., 100 Cummings Center, Beverly, MA 01915-6101

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Isobaric interferences in environmental samples can compromise trace LC-MS analysis of polar organic chemicals when using single quadrupole instruments in selected ion monitoring (SIM) mode. HPLC-MS/MS with triple quadrupole instruments in multiple reaction monitoring (MRM) mode is the conventional approach for increasing selectivity and improving sensitivity. Time-of-flight (ToF) MS offers an alternative approach with higher mass resolving power and full spectral sensitivity. Extracts from STP effluent were evaluated for PPCPs using three different types of Micromass, Inc. mass spectrometers: a single quadrupole (LCZ™), a triple quadrupole (Quattro LC™), and an orthogonal acceleration ToF instrument (LCT™). HPLCToF-MS provided significant S/N improvement over SIM

© 2003 American Chemical Society

109

In Liquid Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS; Ferrer, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


110 analysis for each of the analytes detected and approached S/N values afforded by M R M analysis. In addition, the ToF was also able to identify non-target analytes based on accurate mass measurements and associated elemental composition calculation as illustrated by the detection and confirmation of a polyethylene glycol (PEG) homologous series in STP influent. Limited dynamic range on the ToF as compared to the quadrupole was partially corrected for using digital dead time correction (DDTC).

Introduction Over the past decade, the combination of high performance liquid chromatography and with mass spectrometry (HPLC-MS) has proven to be an important tool for trace analysis of polar organic contaminants in the aquatic environment. A recent development within the field of HPLC-MS has been the emergence of time-of-flight (ToF) mass analyzers with atmospheric pressure ionization interfaces (including electrospray and atmospheric pressure chemical ionization). ToF mass spectrometers operate by accelerating ions into a fieldfree drift region of a fixed path length. The time required to traverse the flight tube and strike a detector is precisely measured and related to the mass-tocharge ratio (m/z). The square of the flight time is proportional to m/z of a particular ion. Modern ToF instruments display superior resolving power than quadrupoles and ion traps along with faster data acquisition rates than quadrupoles and sector instruments. These attributes facilitate the routine measurement of accurate masses, even from narrow chromatographic peaks. Advantages inherent to ToF mass spectrometers also include a wide mass range and full spectral sensitivity. These features make HPLC-ToF-MS instruments attractive for environmental analyses as analytes are often detected at trace levels in a complex matrix. The high sensitivity of HPLC-ToF-MS allows for trace-level quantitation (ppt) and the enhanced resolution in comparison to quadrupole instruments make it better suited for resolving isobaric spectral interferences. Also, since ToF instruments continually acquire data for all ions across a given mass range, non-target analytes can be investigated without compromising sensitivity. In the past, detection and quantification of polar organic contaminants in environmental samples have been most often performed with scanning MS



Ill instruments such as quadrupoles. Single quadrupole HPLC-MS systems operated in single ion monitoring (SIM) mode are robust and sensitive for targeted analyses in relatively clean matrices. This instrumental configuration has been the workhorse for many environmental applications but is susceptible to isobaric interferences (i.e. the low resolving power of the instrument cannot distinguish analytes from interferences with nominally identical m/z). This lack of specificity can lead to decreased signal-to-noise (S/N) ratio, (thereby degrading sensitivity) and an incorrect estimation of analyte concentration (1). Despite these limitations, robust environmental HPLC-MS applications have been developed using single quadrupole mass spectrometers. For example, LCMS has been used in the investigation of antimicrobials in groundwater and surface water (2), polyethylene glycol (PEG) in environmental waters (3), as well as alkylphenol ethoxylates and steroid hormones in wastewater impacted water (4,5,6). Other applications of HPLC-MS include investigation of pesticides and metabolites in surface and groundwater (7,8) and characterization of alcohol ethoxylate biodégradation intermediates (9). Triple quadrupole (MS/MS) instruments operated in multiple reaction monitoring (MRM) mode are less affected by isobaric interferences and have been the analytical gold standard for quantitative environmental analysis of polar organic contaminants. For example, HPLC-MS/MS has been used in the study of neutral pharmaceuticals (10,11) antibiotics (11,12) and X-ray contrast agents (13) present in aqueous media (e.g. groundwater, river water, wastewater, etc.). Although HPLC-MS/MS affords superior selectivity and sensitivity, considerable method development is required (e.g. determining M R M transitions for each analyte and optimizing instrument parameters for each separate transition). More importantly, quadrupole instruments operating in SIM or M R M mode will only detect target ions, and increasing the number of target analytes or transitions will reduce instrument sensitivity. HPLC-ToF-MS provides a powerful alternative or valuable compliment to traditional HPLC-MS or HPLC-MS/MS approaches in environmental contaminant analyses. Initial studies include accurate mass confirmation and analysis of pesticides and metabolites in groundwater (14,75), and surface water (16), as well as screening and identification of unknown contaminants in surface water (17). However, to date there have been no direct comparisons of HPLCToF-MS to quadrupole-based MS for the analysis of trace contaminants in environmental samples.

Scope of Study In the present study, three mass analyzers were compared for the analysis of pharmaceuticals and personal care products (PPCPs) in wastewater treatment



112 plant (WWTP) effluent: an HPLC-ToF-MS instrument, a single quadrupole MS operated in SIM mode, and a triple quadrupole MS operated in M R M mode. A set of 22 frequently detected PPCPs was targeted for this work. Analyses were performed using nearly identical chromatography and electrospray ionization conditions (identical LC columns, MS ion sources, etc.) so that the mass spectrometric analyses could be directly compared. The relative performance and utility of the three instruments with respect to useful sensitivity, selectivity, and dynamic range were assessed. Furthermore, the ability of HPLC-ToF-MS to perform trace-level identification of unknown peaks and confirmation of target and non-target analytes was evaluated.

Methods

Sample Collection and Preparation Wastewater samples (lOOmL influent and 4L effluent) were collected on March 22, 2002 from a tertiary WWTP using solvent rinsed glass bottles. At the same time, a field blank (4L of Milli-Q purified water) was prepared in an identical sampling container. Effluent was immediately filtered under vacuum upon return to lab using Whatman glass fiber filters (0.7μτη particle retention). Two 500mL aliquots offilteredeffluent and 500mL of the field blank were each transferred to separate 1L solvent-rinsed Boston Round bottle and 50 ng of surrogate standard was added (ΙΟμΙυ of a 5ng^L standard of C-phenacetin in H 0). Sample extraction was based on a recently published method for the extraction of PPCPs from surface and groundwater (11). Briefly, samples were extracted at approximately 15 mL/min using Oasis HLB SPE cartridges (6cc, 500mg sorbent) that had been conditioned with 2x3mL aliquots of methanol, followed by 2x2mL aliquots of MilliQ H 0. Upon completion of the extraction, SPE cartridges were washed with lmL of 5% methanol in MilliQ H 0 and dried under vacuum for 1 hour. PPCPs were eluted under vacuum using 2x3mL aliquots of methanol followed by 2x2mL aliquots of 0.1% TFA in methanol. Eluent was collected in glass test tubes, and the two separate extracts were combined, mixed, and again split in half to ensure an identical matrix. The samples were then evaporated to dryness under a gentle N stream, reconstituted with 1 mL of HPLC starting mobile phase containing 50 ng internal standard ( C-caffeine), and ultrasonicated for 15 min to ensure complete solubilization and homogenization. B

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Samples were then filtered using Millipore Ultrafree-MC 0.45μπι centrifugal filters and transferred to 1 mL HPLC injection vials. The two 100 mL influent samples were vacuum filtered using Whatman glass fiber filters (0.7μπι particle retention) and transferred to lOOmL round bottom flasks which had been baked at 400°C and solvent rinsed. No preconcentration or clean-up step was employed prior to analysis.

Instruments and LC Conditions Three Micromass, Inc. mass spectrometers were used in the present study: a single quadrupole MS (LCZ™) with an Hewlett Packard 1100 LC, a triple quadrupole MS (Quattro LC™) with a Hewlett Packard 1100 LC, and a ToF-MS (LCT™, equipped with a 4.6 GHz time-to-digital converter) with a Waters 2695 LC. LC conditions (mobile phases, gradient, etc..) for each instrument were similar as follows: mobile phase " A " was 5 mM formic acid/5 mM ammonium formate buffer (pH = 3.7) in MilliQ water and mobile phase " B " was acetonitrile. 10 of sample was injected and separated using a Betasil C (Keystone Scientific) 150 χ 2.1mm, 3 μιη analytical column with a similar guard column at a flow rate of 0.2 mL/min. Initial solvent composition was 95:5 (A:B) and a linear gradient was employed with a final solvent composition of 10:90 (A:B) at 28 min. Following the gradient, the initial solvent conditions were restored over a 5 minute ramp and the column was allowed to reequilibrate for an additional 12 minutes. For quantitation, a six-point calibration curve (from 1 ng/mL to 500 ng/mL) was constructed for each analyte on each instrument with analyte response normalized to the internal standard. 18

Single-quadrupole MS conditions +

Analytes were detected in SIM mode as (M+H) ions. The sample cone voltage in the electrospray source was held at 31 volts in order to limit fragmentation. Eleven SIM masses were monitored during the first 17 minutes. Ten SIM masses were monitored for the last 11 minutes. Table 1 includes a list of analytes and their corresponding SIM ion m/z values.

Triple-quadrupole MS Conditions Detection of analytes was performed using M R M mode. The sample cone voltage in the electrospray source was optimized for minimal fragmentation and


114 maximum overall sensitivity (34 volts). Eleven M R M transitions were monitored for the first 16 minutes, followed by an additional 11 M R M transitions over the last 12 minutes. Table 1 includes precursor-product MS/MS transitions as well as CED collision energies for each analyte.


L C T Conditions The ToF analysis was conducted in ESP+ mode with a 1.050 second cycle time (1 sec acquisition time and 0.05 sec post-acquisition delay). The selected m/z range was 100 to 1000 Da. The instrument was operated at a mass resolution of 6800 (FWHM), and was externally mass calibrated using polyalanine (Sigma #P9003). Five ng/mL Leucine enkephalin ((M+H) = 556.2771) was used as a lock mass (to compensate for drift of the external calibration) and was added post-column at a flow rate of 1 μί/ιηΐη. This delivery rate was optimized to ensure that the leucine enkephalin signal would not saturate the detector (ion counts per scan) when the mobile phase was at its highest organic content and ionization/desolvation efficiencies were highest. Data files were internally mass calibrated (vs. the leucine enkephalin lock mass) using the manufacturer's all file accurate mass measure (AFAMM) software process. The resulting files were used in the successive data analyses. Accurate mass measurements were used in conjunction with chromatographic retention time for analyte confirmation. Table 1 lists calculated exact masses for each of the analytes detected. +

Results and Discussion

Chromatography Figure 1 shows a base peak intensity (BPI) chromatogram resulting from an HPLC-ToF-MS analysis of a 50 ng/mL calibration. A base peak intensity chromatogram plots the intense signal from each mass spectrum on a chromatographic time scale. Peaks were generally well separated with minimal tailing, and in general, chromatography was reproducible among instruments. Relative retention times did not vary significantly within or among the


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Table I: Mass analyzer parameters for sample analysis 1M


LCZ Analyte 13C-caffeine 13C-phenacetin Acetaminophen Antipyrine Caffeine Carbamazepine Cimetidine Cotinine Diltiazem Diphenhydramine Erythromycin Fenofibrate Fluoxetine Metformin Nifedipine Paraxanthine Ranitidine Salbutamol Sulfamethoxazole Trimethoprim Warfarin

SIM mass 197.8 180.8 151.8 188.8 194.8 236.8 252,8 176.8 414.9 255.9 734.1 361.1 309.9 129.8 347.0 180.8 314.9 239.9 253.8 290.9 308.9

iM

Quattro II Collision MRM energy (v) transition 22 197.8>139.7 21 180.9>109.8 15 151.7>109.7 26 188.9>55.8 22 195.0>137.7 236.8>193.8 19 253.0>158.7 19 24 176.7>79.7 414.8>177.7 27 255.7>166.7 11 734.5>576.2 25 18 361.1>232.8 310.0>147.7 10 21 129.8>70.8 10 347.1>315.0 180.7>123.8 23 27 315.0>175.7 16 239.9>147.7 21 253.8>155.7 27 291.0>229.9 309.2>162.7 20

LCflM Calculated M+l (Da) 198.0984 181.1058 152.0711 189.1028 195.0882 237.1028 253.1235 177.1028 415.1691 256.1701 734.4690 361.1206 310.1418 130.1092 347.1243 181.0725 315.1491 240.1599 254.0599 291.1457 309.1127


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instruments. Absolute retention times varied only slightly between the single quadrupole and triple quadrupole instruments as they used nearly identical HPLC systems. However, absolute retention times in the HPLC-ToF-MS analysis (Waters HPLC system) were shorter. This was likely the result of differences between the Waters and Hewlett Packard HPLC systems, as well as slight differences in column temperature. Relative analyte responses among instruments were similar, indicating that electrospray conditions were indeed relatively constant for the three instruments considered here.

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TOF MS ES+ BPI 2.70e3

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iTime 20.00 25.00 Figure I. Base peak indexed (BPI) chromatogram of calibration solution containing 50 ng/mL of each PPCP

Comparison of Instrument Sensitivity and Selectivity For individual PPCP measured in wastewater effluent extract, the useful sensitivity and selectivity of analysis depended upon several factors. The nature of the analyte (m/z, ionization efficiency, chromatographic behavior including peak width and retention time), properties of the sample matrix, and ion transmission/detection properties of each instrument all influenced instrumental analysis to varying degrees. Under the present conditions, the single quadrupole instrument was the least sensitive for nearly all of the PPCPs due to greater isobaric interferences and baseline noise in complex sample matrices. In general, the triple quadrupole was the most sensitive due to the selectivity and noise reduction afforded by MS/MS. The overall sensitivity of the HPLC-ToF-



117 MS operated in accurate mass mode was much better than that afforded by the single quadrupole in complex mixture analysis and often approached that obtained by the triple quadrupole. This performance was a result of the increased selectivity (reduction of isobaric interferences) afforded by the higher resolving power of the ToF analyzer. The results obtained for diltiazem in wastewater effluent extract illustrate the relative analytical performance of the three instruments (Figure 2). Peak-topeak signal to noise (S/N) ratios were calculated for the SIM and M R M analyses in addition to the ToF accurate mass chromatogram. These chromatograms are shown in Figure 2 for diltiazem in wastewater effluent, including a section of expanded baseline, as well as corresponding S/N values. SIR of 10 Channels ES+ 414.9 1.09e6

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Figure 2. Diltiazem response on single quadrupole (A), triple quadrupole (B), and ToF (C): note sectional baseline enhancement scales The complex matrix present in the wastewater effluent extract caused greatly enhanced signal background and isobaric noise in the single quadrupole



118 HPLC-MS analysis. In this case, the baseline accounted for a significant portion (about 15%) of the instrument response throughout the illustrated portion of the chromatogram. Accordingly, the lowest signal S/N was obtained from the single quadrupole SIM analysis. Several distinct peaks other than that produced by diltiazem peak were also present. These peaks were likely due to the presence of partially or completely chromatographically resolved compounds sharing the same nominal mass with diltiazem, which were inherently monitored by the relatively wide (~lDa) SIM window for this compound. Thus, SIM was found to be neither sufficiently selective nor sensitive for the present analysis of PPCP compounds in wastewater effluent. The triple quadrupole MS/MS analysis was the most selective and sensitive. It produced the highest S/N ratio and completely resolved diltiazem from other isobaric interferences. In the case of the triple quadrupole, the noise reduction was due to the inherent specificity of monitoring specific precursor to product ion transitions during the M R M process. Isobaric compounds are unlikely to have identical CED MS/MS transitions and therefore do not produce an instrument response in M R M mode. The accurate mass chromatogram of diltiazem obtained from HPLC-ToFMS analysis was centered about the calculated (M+H) m/z with a 20 mDa window. The resulting S/N was a marked improvement over the single quadrupole SIM S/N and begins to approach that obtained by MS/MS. The improvement over the SIM analysis can be attributed to the high mass accuracy of the ToF analyzer. Where any analyte within approximately 0.5 Da produced a signal in SIM, only analytes within 0.010 Da registered a signal in the ToF accurate mass chromatogram. In addition, most of the isobaric interferences observed during SIM were not apparent in the ToF analysis. This was a result of considerably higher mass resolution obtained by the ToF, which was in most cases capable of eliminating nominally isobaric spectral mass contaminants. In general, although the ToF was usually less sensitive than the triple quadrupole instrument for analysis of targeted PPCPs in wastewater, it outperformed the single quadrupole in complex mixtures while providing an additional level of confidence in analyte identification via accurate mass measurement. Figure 3 further illustrates the specificity of accurate mass analysis using ToF-MS. A l l five reconstructed ion chromatograms are from the same ToF analysis of diltiazem in the wastewater effluent sample, however each is centered on the calculated exact mass with mass windows of varying size. Thus, when a small mass window is used, the instrument is selective and produced a higher S/N (Figure 3A). As the mass window is widened, the instrument detects isobaric interferences for diltiazem, resulting in a noisier baseline and decreasing S/N values. The smallest window size achievable is practically limited by the average mass measurement error of the instrument. In the case of the LCT™ presently investigated, it was found that a 0.20 mDa window was optimal. Figure 3E represents 1 Da mass window and can be regarded as a simulation of an instrument operating at unit mass resolution. +


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Figure 3. Selectivity and sensitivity improvements in HPLC-ToF-MS analysis of diltiazem in wastewater effluent with narrowing mass window (note the improvement in S/N with decreasing mass window)


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Quantitation of PPCPs in wastewater effluent The analytical performance of the three MS instruments compared in the present work was observed to significantly impact their relative abilities to perform reliable quantitation of PPCPs in wastewater effluent. Table II lists the concentrations determined for the PPCPs detected in the wastewater effluent extract as well as accurate mass measurements and corresponding errors obtained from the ToF analysis. Concentrations obtained from SIM analysis using the single quadrupole instrument were consistently more than a factor of two different than concentrations estimated from MS/MS or ToF analyses. In the cases where a higher concentration was observed from the single quadrupole analysis (as compared to the MS/MS or ToF values), the instrument response was likely influenced by co-eluting isobaric interferences leading to an overestimation of analyte concentration. For analytes detected using the single quadrupole in lower concentrations than estimated by MS/MS or ToF, it is possible that poor chromatographic resolution from a noisy baseline contributed to observed inaccuracies. It is unlikely that these discrepancies were a result of matrix enhancement or suppression as nearly identical chromatography and ionization sources were used for each instrument. The ability of the ToF mass analyzer to resolve interferences leads to more reliable determinations of analyte concentrations (closer to the results achieved using MS/MS) in comparison to the results obtained from the single quadrupole MS. With few exceptions, concentrations determined from HPLC-ToF-MS were within a factor of two of those obtained using the triple quadrupole. In the case of sulfamethoxazole, it is not clear why the ToF seems to have underestimated the concentration relative to the MS/MS results. In the cases of caffeine and paraxanthine, however, the C isotope peak from a co-eluting compound with a mass 1 amu lower than the analytes caused an overestimation of analyte concentration (Figure 4). Although the interferences are not resolved for either example, the ratios of the 194 to 195 and 180 to 181 peaks suggest a significant contribution from the C isotope from the lower mass compounds. Consequently, the accurate mass calculations for the target peaks reflect the influence of the suspected isobaric interferences (Table II). The associated mass error is significantly higher than the 2.0 mDa accuracy specified by the manufacturer. Thus, the accurate mass measurement capability of the ToF instrument can sometimes provide valuable evidence for an isobaric interference. Such information was not available using either single or triple quadrupole instruments. 13

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121 Table II: PPCP Quantification using different mass analyzers


Compound*

PPCP concentrations in effluent ToF accurate mass (ng/L) Mass Accurate Quattro error LCT™ LCZ™ mass (Da) LC™ (mDa) -20.0 195.1082 43.6 109 210 237.1027 0.1 194 888 119 0.5 253.1230 193 240 88.6 1.2 177.1016 10.2 22.0 nd** 415.1685 0,6 72.4 52.4 21.8 0.3 314 256.1698 117 361 307 nd nd nd nd 19.9 0.5 130.1087 nd 243 261 181.0956 -23.1 154 478 35.3 315.1528 -3.6 184 91.0 51.4 0.2 240.1597 9.44 35.6 nd 254.0604 -0.5 458 2420 1570 0.5 291.1452 19.2 130 105 9.78 nd nd -

Caffeine Carbamazepine Cimetidine Cotinine Diltiazem Diphenhydramine Erythromycin Fenofibrate Metformin Paraxanthine Ranitidine Salbutamol Sulfamethoxazole Trimethoprim Warfarin

*Only those PPCPs that were detected in the STP effluent extract are listed. Not detected e

100-

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180.0954

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Theoretical caffeine (M+1f= 195.0882

181.0956 195.1082

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Figure 4. Caffeine and paraxanthine mass spectra illustrating interfering ^C isotope peaks derived from intense spectra peah ~7 Da smaller than the analytes


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Target and non-target analysis of environmental contaminants in complex samples using accurate mass HPLC-ToF-MS Three distinct advantages offered by ToF-MS include the ability to collect data across a wide mass range without sacrificing sensitivity, the ability to resolve interferences away from signals of interest, and mass measurement accuracy adequate for the estimation of elemental composition. The accurate mass measurements for the target analytes in the wastewater effluent involved a single point correction of the base calibration (to compensate for slight drift of the calibration due to temperature fluctuations in the flight tube and instabilities of the power supplies) utilizing a reference compound, or lock mass. Table II includes the measured masses for those compounds in addition to their mass error relative to calculated exact masses (listed in Table 1). Mass measurement error was determined as the difference between the calculated and measured (M+H) . For most of the target analytes, this error was very low, usually below 1 mDa. In these cases, elemental composition calculated by the instrument software provided an additional means of analyte confirmation. As previously mentioned, in the cases where the mass error was highest, it appeared that an unresolved peak had interfered with the mass measurement of the target compound (Figure 4). The resolving power, accurate mass measurement capability, and full spectral sensitivity also make LC-ToF-MS attractive as a tool for identifying non-target compounds in complex environmental matrices. In principle, if masses can be measured with sufficient accuracy, it is possible to assign unique elemental compositions to peaks observed during the course of an analysis. In practice, a mass measurement within 2 mDa gives rise to a short list of elemental compositions to consider. In combination with other information such as calculated isotope ratios and chromatographic retention times of standards, it is obvious that HPLC-MS employing accurate mass measurement holds great promise for rapid qualitative analysis of "unknown" environmental mixtures. As an example, a filtered wastewater influent sample was analyzed using the HPLC-ToF-MS system directly without an extraction/cleanup step. Utilizing a presentation of peak intensity as a function of m/z and retention time, patterns of sequentially eluting homologous series separated by 44 Da were identified. One series displayed m/z values consistent with protonated polyethylene glycol (PEG) molecules. Extracted ion chromatograms were created for representative members of the series, and accurate mass measurements confirmed elemental compositions of the PEGs. Figure 5 shows narrow window the accurate mass chromatograms of selected members of the observed PEG homologous series as well as the mass measurement error. It should be noted that identification of the PEG series in the present example was greatly facilitated by the polymeric nature of the material (i.e. the presence of a +


123 distinct pattern of repeating peaks in the observed mass spectrum). This feature made it relatively easy to find the series visually in the display of the HPLC-MS dataset. General "non-target" analyses will likely require sophisticated data processing software tools for rapidly and reliably interpreting data from accurate mass HPLC-ToF-MS runs.

TOF MS ES+ PEG5: 239.1481 error = -1.4 mDa

100 Downloaded by UNIV OF GUELPH LIBRARY on September 2, 2012 | http://pubs.acs.org Publication Date: May 20, 2003 | doi: 10.1021/bk-2003-0850.ch007

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% '

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Figure 5. Accurate mass chromatograms ofselected PEG oligomers in wastewater influent: PEG ethoxy-chain length and corresponding m/z are shown at right, top; mass error (mDa) is shown at right bottom


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Lock mass as suppression indicator One problem associated with electrospray ionization is suppression or enhancement of signal in the presence of co-eluting sample matrix. Often, components co-eluting with an analyte can preferentially occupy surface sites of the electrosprayed droplets, or compete for available charge, thereby decreasing analyte signal intensities {18,19). In the present case, the signal obtained from the lock-mass used to determine accurate mass (leucine enkephalin) inadvertently, but advantageously, provided a useful record of matrix effects. Because the lock mass was added post-column (just prior to the electrospray source), comparison of the response from a "clean" sample (e.g. a calibration solution) to that of a matrix-rich sample provided an indication of the presence and degree of analyte signal suppression. Figure 6 shows two overlaid leucine enkephalin response plots; one from a calibration solution, and the other from a wastewater effluent sample. Aside from the area between 12 and 17 minutes, there are few differences in the quantitative responses of the lock mass in the two analyses. During the period between -12 and 17 minutes, the lock mass signal from the effluent analysis appears to be substantially suppressed relative to that measured in the analysis of the calibration solution. Consequently, it is likely that analytes eluting in this window may also have been subject to signal suppression. This added information provided a valuable internal check on the efficiency of electrospray ionization in the analysis of a complex mixture. Thus, post-column lock mass infusion (which is commonly employed for accurate mass studies in HPLC-ToF-MS) can serve as a useful secondary purpose as an indicator of matrix-induced signal suppression events often associated with HPLC-ESI-MS analyses of environmental samples.

TOF MS ES+ 556.277 0.02Da 1.78e3

10O,

Lock mass response in calibration solution Lock mass response in STP effluent extract 0.00

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Figure 6. Lock mass (leucine enkephalin) response in a calibration solution and in the wastewater effluent extract showing evidence of matrix-induced signal suppression.


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The digital dead time correction algorithm and its effect on ToF-MS dynamic range The majority of commercially available HPLC-ToF-MS systems employ a time-to-digital (TDC) converter to process signals associated with individual ion arrival times at a multichannel plate detector. There is a finite time interval (4 nsec on the present instrument) required between ion detection events, which limits the linear dynamic range of the instrument. As the TDC systems become saturated (arrivals of nearly the same m/z ions at less than 4 nsec intervals), there occurs both a loss of linearity in detector response (Figure 7), and a shift in the signal to lower m/z, resulting in poor mass accuracy. In the present work, it was found that the effective linear dynamic range of the ToF-MS was between two and three orders of magnitude. This is significantly lower than the dynamic range typically observed on quadrupole instruments (typically >4 orders of magnitude). In actuality, this limitation is one of the most important drawbacks of using HPLC-ToF-MS for quantitative measurements of environmental contaminants. Software corrects moderate levels of detector saturation and the associated mass shifts with an algorithm called digital dead time correction (DDTC). This technique allows limited correction of the mass centroid (and peak intensity) of acquired spectra by a back-calculation algorithm triggered when the number of ion counts per second exceeds a user-defined "dead-time saturation" value. Figure 7 illustrates the effect of DDTC processing on the quantitative calibration dynamic range of cotinine acquired with the HPLC-ToFMS system.

600 Cotinine concentration (ng/mL)

Figure 7. Effect of digital dead-time correction (DDTC) on ToF dynamic range for cotinine


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Conclusion The present work illustrates that HPLC-ToF-MS provides powerful and complimentary information to that obtained by LC-MS/MS in the analysis of polar organic contaminants in a complex environmental matrix. Figure 8 provides a comparison of the relative advantages and disadvantages of the three types of instruments used when applied to environmental analysis. Due to higher mass resolving power, ToF-MS can provide S/N improvements when compared to single quadrupole analyses of complex samples. A n important benefit of accurate mass determinations using ToF-MS is useful information about elemental compositions, which can confirm or rule out potential molecular formulas. The full spectra sensitivity of ToF-MS provides an important advantage for conducting survey or discovery based analyses in comparison to scanning MS instruments where sensitivity can be limited even when scanning over a narrow mass range. Consequently, the strengths of HPLC-ToF-MS appear well suited for challenges inherent to the analysis of polar organic contaminants and their metabolites in the aquatic environment. Sensitivity LCToF-MS

Selectivity +++ (accurate mass)

Identity confirmation

Dynamic range

Analyte discovery

cost

-H-+

+ +++ $$ (elemental (102-1Q3) composition) LC-MS + +++ + + + $ (nominal (>10 ) (SIM) mass) LC++ 4-H++ +++ ++ $$$ MS/MS (product (>104) (MRM) spectra) Figure 8. Relative comparison of evaluated HPLC-MS systems ++

4

Acknowledgements This work was supported by an EPA STAR Grant (#R-82900701-0). Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through grant/cooperative agreement (#R-82900701-0) to Bruce Brownawell, it has not been subjected to the Agency's required peer and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred.


127

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MS for Sensitive and

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