MS

Anal. Chem. 2005, 77, 4453-4458

Analysis of Perchlorate in Water by Reversed-Phase LC/ESI-MS/MS Using an Internal Standard Technique Yongtao Li* and Ed J. George

Environmental Health Sciences, Underwriters Laboratories Inc., 110 South Hill Street, South Bend, Indiana 46617

A new method was developed for the analysis of perchlorate in water by using reversed-phase liquid chromatograhy/electrospray ionization-mass spectrometry/ mass spectrometry (LC/ESI-MS/MS) in the negative ESI mode. Selective and sensitive perchlorate detection was obtained by monitoring the 35ClO4- f 35ClO3- and 37ClO - f 37ClO - mass transitions. The 35ClO - f 4 3 4 35ClO - transition was quantitated against the internal 3 standard oxygen-labeled sodium perchlorate (NaCl18O4). Sample pretreatment for the removal of major common anions and dissolved metal ions along with internal standard quantitation sufficiently compensated for ion suppression caused by the matrix. The 37ClO4- f 37ClO3transition was examined to provide additional specificity. The method sensitivity, accuracy, and precision were investigated by analyzing fortified blank samples, field samples, and performance evaluation samples. The results (1.01-13.5 µg/L) for the proficiency evaluation samples differed from the certified values (1.04-14.1 µg/ L) by 3-18%. The developed reversed-phase LC/ESI-MS/ MS method was rapid, accurate, and reproducible. The calculated method detection limits were 0.007 µg/L for deionized reagent water and 0.009 µg/L for synthesized reagent water, respectively. The minimum reporting limit was conservatively set to 0.05 µg/L. Perchlorate is persistent in the environment due to its high solubility and extreme stability in aqueous media. Perchlorate contamination in drinking water supplies, agriculture irrigation, and food supplies is of concern due to possible adverse impact of perchlorate on human health at relatively low concentrations.1-3 The largest source of perchlorate contaimination is from the manufacture of rocket fuel. It has been detected in ground, surface, and drinking water sources in many states in the United States.4,5 Standards for perchlorate in drinking water have been * To whom correspondence should be addressed. E-mail: yongtao.li@ us.ul.com. Fax: 574-233-8207. (1) Urbanky E. T.; Shock, M. R. J. Environ. Manage. 1999, 56, 79-95. (2) Clewell, R. A.; Merrill, E. A.; Yu, K. O.; Mahle, D. A.; Sterner, T. R.; Mattie, D. R.; Robinson, P. J.; Fishert, J. W.; Gearhart, J. M. Toxicol. Sci. 2003, 73, 235. (3) Kirk, A. B.; Smith, E. E.; Tian, K.; Anderson, T. A.; Dasgupta, P. K. Environ. Sci. Technol. 2003, 37, 4979-4981. (4) Hogue, C. Chem. Eng. News 2003, 37. (5) Okamoto, H. S.; Rishi, D. K.; Steeber, W. R.; Baumann, F. J.; Perera, S. K. J. Am, Water Works Assoc. 1999, 91, 73-84. 10.1021/ac0500986 CCC: $30.25 Published on Web 06/01/2005

© 2005 American Chemical Society

established in many states. The lowest action level is at 1 ppb.4 Perchlorate has been also found in soils,6 fertilizers,7 plants,8 and milk.3 Ion chromatography (IC) coupled with conductivity detection (CD) is a common technique used for perchlorate analysis in water, which provides a detection limit of ∼1 ppb and a minimum reporting limit (MRL) at low-ppb levels, depending on the complexity of the sample matrix and the sample pretreatment.5,9 Because CD responds to any species with sufficient conductivity, the lack in specificity will result in a need for confirmatory testing. Severe signal suppression is seen for samples that contain common anions, particularly sulfate and chloride at much higher concentrations than perchlorate. The presence of these anions increases the likelihood of false negative results. False positive results caused by other unknown nontarget contaminants are often experienced with IC/CD. The effects of common anions can be reduced by using conductivity suppression and sample preconcentration techniques. Tian10 developed an IC/CD method coupled with automated online preconcentration and preelution. A recovery of 92% was obtained for perchlorate at 25 ppb in the test matrix containing 2000 mg/L each of SO42-, Cl-, and CO32-. Hedrick11 reported that a six-port switching valve placed before a conductivity suppressor could divert the IC separation column eluate from the suppressor as well as the MS system, which significantly reduced the background noise and sample cone fouling resulting from the SO42eluting from the IC column. The direct analysis of perchlorate by using electrospray ionization (ESI) MS and MS/MS improved the sensitivity and selectivity in the analysis through characteristic mass-based determination.12-14 However, ion suppression is a well-documented (6) Winkler, P.; Minteer, M.; Willey, J. Anal. Chem. 2004, 76, 469-473. (7) Susarla, S.; Collette, T. W.; Garrison, A. W.; McCutcheon, S. C. Environ. Sci. Technol. 1999, 33, 3469-3472. (8) Ellington, J. J.; Wolfe, N. L.; Garrison, A. W.; Evans, J. J.; Avants, J. K.; Teng, Q. Environ. Sci. Technol. 2001, 35, 3213-3218. (9) Hautman, D. P.; Munch, D. J.; Eaton, A. D.; Haghani, A. W. U. S. Environmental Protection Agency Method 314.0, Revision 1.0, 1999. (10) Tian. K.; Dasgupta, P. K.; Anderson, T. A. Anal. Chem. 2003, 75, 701706. (11) Hedrick, E.; Munch; D. J. Chromatogr., A 2004, 1039, 83-88. (12) Magnuson, M. L.; Urbansky, E. T.; Kelty, C. A. Anal. Chem. 2000, 72, 2529. (13) Koester, C. J.; Beller, H. R.; Halden, R. U. Environ. Sci. Technol. 2000, 34, 1862-1864. (14) Urbansky, E. T.; Magnuson, M. L.; Freeman, D.; Jelks, C. J. Anal. Atm. Spectrosc. 1999, 14, 1891-1866.

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problem associated with ESI,12,15 which may adversely affect the accuracy and precision of perchlorate determination in flow injection analysis without adequate chromatographic separation. Severe ion suppression caused by common anions leads to differences in calibration plot slopes.12,13 Sample dilution is typically used to minimize ion suppression without increasing analytical cost and complexity. With a 10-fold dilution, ESI-MS/MS provides a detection limit of ∼0.5 ppb for perchlorate analysis.13 Other approaches used to enhance the selectivity and sensitivity for perchlorate analysis also include ion pair liquid-liquid extraction,12 standard additions,14 and stable association complex formation.13 Ells16 developed an ESI-FAIMS/MS method, which provided a detection limit of 0.05 ppb for perchlorate in a relatively clean matrix of tap water. A number of laboratories have coupled IC with MS or MS/MS to increase detection specificity and sensitivity and to reduce matrix effects. The isotopic ratio of 35ClO4 f 35ClO3(m/z 99 to 83) transition to 37ClO4- f 37ClO3- (m/z 101 to 85) transition is near the ratio of 35Cl f 37Cl for natural isotopic abundances and provides additional analyte identification specificity and confidence in the ESI-MS/MS analysis of perchlorate.6,13 18O-enriched perchlorate (Cl18O -) is an effective internal 4 standard. Because of the similarity of perchlorate and the selected IS in the processes of chromatographic separation and ESI, this IS can provide the highest degree of accuracy when instrument sensitivity drift and ion suppression occur. Hedrick11 obtained a detection limit of 0.02 ppb for perchlorate analysis in distilled, deionized reagent water. Krynitsky17 reported that the limits of quantitation for perchlorate were 0.5 µg/L in bottled water, 1.0 µg/kg in lettuce, 2.0 µg/kg in cantaloupe, and 3.0 µg/L in milk. Another approach to minimize the ion suppression for perchlorate analysis is to pretreat samples prior to analysis. Dionex (Sunnyvale, CA) OnGuard cartridges in barium, silver, and hydrogen forms are effective for removing sulfate, chloride, carbonate, and heavy metal ions from water samples. By incorporating the use of these pretreatment cartridges, Winkler6 developed an IC/ESIMS/MS method that provided a detection limit of 0.05 ppb for perchlorate analysis in water. One disadvantage of pretreating samples with these cartridges is that it increases the analytcal cost. In this work, a reversed-phase LC ESI-MS/MS method was studied to achieve rapid, sensitive, and accurate analysis of perchlorate in water. By incorporating the use of Dionex OnGuard cartridges for the removal of common anions sulfate, phosphate, carbonate, and chloride, the reversed-phase LC column under a moderate pH condition was used to separate perchlorate anion from the other common anions that were present in the solution. The effects of common anions on separation and detection were investigated. The method sensitivity, accuracy, precision, and applications to field samples have been investigated. The 18Oenriched perchlorate internal standard was also used to compensate for matrix effects, particularly the ion suppression caused by the presence of common anions. Sensitivity, accuracy, and precision have been improved. (15) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (16) Ells, E.; Barnett, D. A.; Purves, R. W.; Guevremont, R. J. Environ. Monit. 2000, 2, 393-397. (17) Krynitsky, A. J.; Niemann, R. A.; Nortrup, D. A. Anal. Chem. 2004, 76, 5518-5522.

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EXPERIMENTAL SECTION Standards and Reagents. The 1.0 mg/mL perchlorate and other anion standard stock solutions were obtained from Inorganic Ventures, Inc. (Lakewood, NJ) and AccuStandard, Inc. (New Haven, CT). The internal standard (IS) was oxygen-labeled sodium perchlorate (NaCl18O4) that was provided by the United States Environmental Protection Agency (USEPA). The IS should have 18O enrichment of greater than 90%. However, the IS at 10 µg/L was also verified prior to each analysis. There were no detectable peaks at m/z 83 resulting from 35Cl16O4 f 35Cl16O3- transition and at m/z 85 resulting from 37Cl16O4- f 37Cl16O3- transition. Highpurity methanol and acetic acid were obtained from Fisher Scientific (St. Louis, MO). Deionized reagent water (18.2 MΩ‚ cm resistance) was obtained from a Milli-Q treatment unit (Millipore, Bedford, MA). All the other neat chemicals, including sodium sulfate, sodium carbonate, potassium phosphate (monobasic), and sodium chloride, were obtained from Fisher Scientific. Sample Collection, Storage, and Preparation. Samples were collected in 100-mL precleaned plastic bottles. No preservation was required.9 According to the literature,6,9,13 perchlorate is stable at room temperature for 50 days or longer. Therefore, the holding time of 28 days was recommended to be consistent with USEPA anion analysis guidelines. Samples were pretreated to eliminate potentially high concentrations of common anions prior to analysis, which was accomplished by eluting an aliquot of an ∼2-mL sample through Dionex (Sunnyvale, CA) barium, silver, and hydrogen OnGuard cartridges in series. Based on the manufacturer-recommended procedures, the sample aliquot was eluted through the cartridges at a speed of ∼1 mL/min. A 1-mL sample was fortified with the IS at a concentration of 10 µg/L prior to analysis. The sample preparation process took ∼2 min for each sample. ESI-LC/MS/MS Conditions. The separation was carried out using an Alliance 2695 HPLC system with a Xterra MSC18 column (2.1 mm × 150 mm, 3.5 µm) (Waters, Milford, MA). A SecurityGuard C18 guard column (2 mm × 4 mm) (Phenomenex, Torrance, CA) was used to protect the analytical column. The mobile phase was an isocratic flow of 90/10 of 0.1% acetic acid/ methanol at a flow rate of 0.25 mL/min. The LC separation was set to 6 min. After processing a sample, the instrument went through a short normal purging and rinsing process and then returned to the initial injection position. No other cycle time was needed because of the use of the isocratic mobile phase. The column temperature was set to 40 °C. The injection volume was 50 µL. Under these conditions, the retention time of perchlorate and the IS was ∼3.8 min. The detection was carried out using a Quattro micro API triple quadrupole mass spectrometer using MassLynx version 4.0 software (Waters). The instrument was calibrated by using an API test kit provided by the manufacturer, which was a sodium iodide and cesium iodide mix. The instrument was run in a negative ion mode and was optimized to obtain sufficient sensitivity and resolution. The key optimized conditions included a 2.5 kV capillary voltage, 45 V cone voltage, 130 °C ion source block temperature, 400 °C desolvation temperature, 750 L/h desolvation gas flow, 50 L/h cone gas flow, 6.0 × 10-3 mbar collision cell gas pressure, and 20 eV collision energy. The LM and HM resolutions were set to 12 for each quadrupole. The data acquisition was set

Figure 1. Selected chromatograms of perchlorate and internal standard in deionized reagent water using 10:90 methanol/0.1% acetic acid as the mobile phase. (a) m/z 101 to 85 for 0.05 µg/L perchlorate, (b) m/z 99 to 83 for 0.05 µg/L perchlorate, (c) m/z 107 to 91 for 10 µg/L IS, and (d) m/z 107 to 89 for 10 µg/L IS.

in a MRM type. Four MRM transitions were monitored, which included m/z 99 to 83, 101 to 85, 107 to 89 (35Cl18O4- f 35Cl18O3-), and 109 to 91 (37Cl18O4- f 37Cl18O3-). The interchannel delay was set to 0.01 s. The dwell time was set to 0.5 s. The data acquisition was set to 6 min. Sample Analysis and Quantification. A new calibration was analyzed with samples on a daily basis. The calibration curve was obtained by analyzing a series of perchlorate standard solutions at 0.02, 0.05, 0.1, 0.5, 1, 5, and 10 µg/L. Each standard solution contained the IS at a constant concentration of 10 µg/L. One measurement was made at each concentration level. Perchlorate was quantified using an internal standard calibration method, which was based on the peak areas for the m/z 99 to 83 transition. The IS was quantified using an external calibration method, which was based on the peak area for the m/z 107 to 89 transition. The mass spectral peaks produced by other transitions were used for confirmation, based on the natural isotopic abundances.6 The internal standard in each analysis provided a quality control of sample pretreatment, instrument performance, and matrix effects. The retention time shifting and peak areas of the internal standard in each analysis were used to check matrix effects, primarily ion suppression. To demonstrate that the instrument was properly calibrated throughout the analysis, a continuing calibration check sample was analyzed at the end of the analysis batch to verify the calibration. An external quality control sample was analyzed to verify the calibration standards. A laboratory method blank was analyzed to demonstrate that there was no carryover from the standards and the sample processing hardware and no interference from the solvents including deionized reagent water. Matrix spike/matrix spike duplicate samples were also analyzed to examine matrix effects. RESULTS AND DISCUSSION Effects of Common Anions. Figure 1 shows the typical chromatograms of 0.05 µg/L perchlorate and 10 µg/L IS in deionized reagent water. The average peak-to-peak S/N ratios were 11 for 35Cl mass transition (m/z 99 to 83) and 1.3 for 37Cl

mass transition (m/z 101 to 85). The peak shape was evaluated using the peak Gaussian factor (PGF), which was calculated using the equation 1.83W0.5/W0.1 (W0.5 is the peak width at half-height and W0.1 is the peak width at tenth height).18 The obtained PGF was 0.93-1.00 for 10 µg/L perchlorate through 18 measurements over a period of three months. To study the effects of common anions on perchlorate analysis, a series of solutions containing 1 µg/L perchlorate, 10 µg/L IS, and common anions at different concentrations were analyzed. The studied common anions included 10-500 mg/L SO42-, CO32-, PO43- as P, and Cl-; 1-30 mg/L NO3- as N; and 0.5-5 mg/L Br-, BrO3-, ClO2-, ClO3-, and F-. Figure 2 shows the chromatograms of 1 µg/L perchlorate and 1 mg/L ClO3-, ClO2-, BrO3-, and Br- anions. Figure 3 shows the chromatograms of 1 µg/L perchlorate, 1 mg/L NO3- as N, 5 mg/L Cl-, 10 mg/L PO43- as P, and 10 mg/L SO42- anions. As shown in Figures 2 and 3, perchlorate was generally separated from Br-, BrO3-, ClO2-, PO43-, and SO42- anions, but partially coeluted with ClO3-, NO3-, and Cl- anions. ClO2- and PO43- anions obviously tailed into the perchlorate peaks. The mass of F- anion was too low to be accurately detected. No peak was detected for CO32- anion. The results indicated that Br-, BrO3-, ClO2-, ClO3-, F-, CO32-, and NO3- anions at the studied concentrations had no significant negative impact on the relative recoveries of perchlorate. Cl-, SO42-, and PO43- were the most problematic anions. They are present in drinking water at relatively high concentrations and could result in retention time shifting as shown in Figure 3, peak broadening, and ion suppression. The absolute recoveries of both perchlorate and the IS decreased with the increase in the concentrations of SO42-, PO43-, and Cl- anions. In addition, Figure 3 also indicates the spectral interferences resulting from SO42and PO43- anions, which could be rationalized as a result of the natural isotopic abundances of 34S (4.4%) and 18O (0.2%). As shown in Figure 3, the following transitions, H34S16O4- f H34S16O3-, H32S18O16O3- f H32S18O16O2-, and H2P18O16O3- f H2P18O16O2-, could cause the spectral interferences (m/z 99 to 83). Therefore, (18) Bassett, M. V.; Wendelken, S. C.; Dattilio, T. A.; Pepich, B. V.; Munch, D. J. U. S. Environmental Protection Agency Method 532, Revision 1.0, 2000.

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Figure 2. Chromatograms of perchlorate at 1 µg/L and common anions at 1 mg/L using 10:90 methanol/0.1% acetic acid as the mobile phase. (A) ClO4- (m/z 99 to 83); (B) ClO3- (m/z 83 to 67); (C) ClO2- (m/z 67 to 51); (D) BrO3- (m/z 127 to 111); and (E) Br- (m/z 79 to 79).

Figure 3. Chromatograms of perchlorate at 1 µg/L and common anions at different concentrations using 10:90 methanol/0.1% acetic acid as the mobile phase. (A) ClO4- (m/z 99 to 83); (B) 1 mg/L NO3- as N (m/z 62 to 46); (C) 5 mg/L Cl- (m/z 35 to 35); (D) 10 mg/L PO43- (m/z 97 to 79); and (E) 10 mg/L SO42- (m/z 97 to 97).

the removal of these anions from the sample was needed to obtain reliable results. The spectral interferences could be also resolved by the use of 37ClO4- f 37ClO3- transition (m/z 101 to 85). Calibration. Eighteen calibration curves ranging from 0.02 to 10 µg/L were used to evaluate calibration linearity. These calibration curves were collected over a period of three months. The linear correlation coefficients (R2) were 0.9974-1.0000 for the individual calibration curves. As shown in Figure 4, the solid line indicates the curve of the mean relative response versus concentration, and the dashed lines indicate a window of (20% about the mean. For all the points except for concentrations at or below the minimum reporting limit (MRL, 0.05 µg/L), the measured relative responses are within this window. The recoveries of perchlorate in the initial calibration standards (ICSs) were 4456 Analytical Chemistry, Vol. 77, No. 14, July 15, 2005

also calculated against the curves. The quality control (QC) criteria for the calibration curves included that the readback recoveries were within (30% for the ICSs at or below the MRL and within (20% for the ICSs at concentrations of higher than the MRL. The isotopic ratios of 35Cl to 37Cl calculated as the MRM peak area ratios were also evaluated using 13 calibration curves collected over a period of three months. As shown in Figure 3, the solid lines indicate the isotopic ratio window within three times the standard deviation (3STD) about the mean at the different concentrations, which can be simply used as the QC criteria of acceptable isotopic ratios. The mean isotopic ratios in the studied concentration range were 2.93-3.17, 96-104% of the isotopic ratio of 35Cl to 37Cl for natural isotopic abundances. All the measured isotopic ratios were within this 3STD window. However, this

Table 1. Method Detection Limit Data (n ) 7)

Figure 4. Calibration curves in deionized reagent water. Individual points: relative responses at each concentration level. Solid line: mean relative responses vs concentration. Dashed lines: mean relative response ( 20% about the mean vs concentration.

Figure 5. Isotopic ratios at different perchlorate concentrations. Y-axis: MRM peak area ratio of 35ClO4 f 35ClO3- to 37ClO4- f 37ClO - transitions. Individual points: isotopic ratios at each concen3 tration level. Solid lines: mean isotopic ratio ( 3STD vs concentration. Dashed lines: theoretical isotopic ratio ( 40% about the theoretical value at the MRL and ( 20% about the theoretical value for concentrations above the MRL.

isotopic ratio QC window might be too wide (>(50%) for concentrations at or below the MRL at 0.05 µg/L and too narrow (