Anal. Chem. 2005, 77, 2475-2481
Analysis of Perchlorate in Human Urine Using Ion Chromatography and Electrospray Tandem Mass Spectrometry Liza Valentı´n-Blasini,† Joshua P. Mauldin,† David Maple,‡ and Benjamin C. Blount*,†
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, and Battelle Memorial Institute, Columbus, Ohio 43201
Because of health concerns surrounding widespread exposure to perchlorate, we developed a sensitive and selective method for quantifying perchlorate in human urine using ion chromatography coupled with electrospray ionization tandem mass spectrometry. Perchlorate was quantified using a stable isotope-labeled internal standard (18O4-perchlorate) with excellent assay precision (coefficient of variation 90%) was obtained from Isotec (Miamisburg, OH). Deionized (DI) water with a specific resistance of 18 MΩ‚cm or greater was used for the study. Synthetic urine was obtained from CST Technologies (Great Neck, NY). Standards Solutions. Standard stock solutions were prepared by dissolving solid ammonium perchlorate in deionized water. This concentrated stock solution was subsequently diluted with DI water to create intermediate stock solutions that were aliquoted and stored at -20 °C until use. A fresh intermediate stock aliquot was used every 2 months. Standard solutions were prepared daily by diluting intermediate stock solutions with synthetic urine to final concentrations that covered the linear range of the assay (0.025-100 ng/mL). Labeled internal standard was prepared in a similar manner, and aliquots of both were stored at 4 °C before use. Quality Control. Analysis of 10 anonymously collected human urine samples was used to set target ranges of two quality control (QC) pools at a low level (5.0 ng/mL, mean perchlorate level for the test population) and at a higher level (75 ng/mL). QC materials were prepared from synthetic urine due to the endogenous content of perchlorate in human urine samples. These QC pools were uniformly mixed and dispensed into polypropylene vials, sealed, and stored at -20 °C until use. QC characterization involved 20 discrete measurements for each QC pool made on 10 separate days by 2 different analysts. These data defined the mean perchlorate concentrations in each QC pool, as well as the 2 standard deviation and 3 standard deviation limits for future precision evaluation. Proficiency Testing. Absolute assay accuracy was verified by the blind analysis of certified perchlorate reference solutions (AccuStandard, New Haven, CT) prepared to final concentration in synthetic urine. Proficiency testing samples were prepared to final concentrations ranging from 0.19 to 72 ng/mL. Proficiency testing samples were run at least two times per year and after any major instrument maintenance. Sample Collection and Storage. Each lot of polypropylene specimen cups, tubes, pipet tips, and autosampler vials was prescreened to confirm no measurable perchlorate contamination. Due to the selectivity and sensitivity of the method either first morning void or spot urine samples were acceptable. Urine samples were aliquoted into polypropylene cryovials and stored frozen until analysis. Urine concentration was assessed by quantifying creatinine using an enzyme-based colorimetric method on a Roche Hitachi 912 Chemistry Analyzer25 and these data used to creatinine-adjust urinary perchlorate data. Sample Preparation. Urine samples were thawed to room temperature and mixed to suspend any particulate material. Urine (0.5 mL) was transferred to an autosampler vial and spiked with 2 ng of labeled internal standard. The sample was diluted with 0.5 mL of DI water and queued for injection into the IC-MSMS system. Chromatography. Ion chromatography was carried out on an IonPac AS16 analytical column (2 × 250 mm; Dionex). Samples were injected using loop injection mode and a 25-µL injection loop. (25) Guder, W. G.; Hoffmann, G. E.; Hubbuch, A.; Poppe, W. A.; Siedel, J.; Price, C. P. J. Clin. Chem. Clin. Biochem. 1986, 24, 889-902.
A 50 mM NaOH eluant was used under isocratic conditions at a flow rate of 0.5 mL/min. The sodium in the mobile phase was removed using a suppressor (Dionex ASRS Ultra II, 2 mm) in external water mode. To ensure appropriate suppressor function, the total conductivity of the postsuppressor eluant was monitored; conductivity greater than 3 µS indicated unacceptable suppressor performance and was remedied by either preparation of new mobile phase or suppressor maintenance. Under these conditions, the retention time of perchlorate was 6 min with a total run time of 10 min. Mass Spectrometry. Following removal of sodium by the suppressor, column eluants were ionized using an electrospray interface to generate and transmit negative ions into the mass spectrometer. To minimize possible fouling of the mass spectrometer, a valve (Valco Instruments, Houston, TX) diverted column eluants except for the time window when perchlorate eluted from the column. The matrix diversion valve was kept in the divert position for the first 4.0 min, after which the flow was directed into the electrospray interface for the next 5.5 min and finally back to the original position for the last 0.5 min of the run. The electrospray probe temperature was maintained at 600 °C when mobile phase passed through the electrospray interface; when mobile phase was diverted from the electrospray interface the probe was set at 200 °C. This procedure helped to minimize fouling of the mass spectrometer by diverting the majority of matrix salts to waste. Mass spectral data were acquired in multiple reaction monitoring mode, cycling between transitions for perchlorate (99 to 83, 101 to 85) and 18O4-perchlorate (107 to 89) with a dwell time of 400 ms for each transition. Nitrogen (highpurity grade) was used as the collision gas. Collision energy and other mass spectral parameters were optimized for maximum transmission of 99 to 83. The mass spectrometer was tuned weekly (or as needed following instrument maintenance) using poly(propylene glycol) as a mass calibrant. Due to the relatively low mass of the analyte ions, the default mass calibration table was modified to include lower mass poly(propylene glycol) species. The mass calibration was evaluated monthly against a mixture of amino acids with molecular weights ranging from 75 to 170 amu. Proper identification of the expected masses confirmed the mass calibration of the instrument. Data Analysis. The identity of the perchlorate peak was confirmed by matching its retention time with the peak produced by the coeluting 18O4-labeled internal standard. All data were evaluated for accuracy of integration and manually reintegrated if necessary. We used the peak area ratio of analyte to stable isotope-labeled internal standard (2 ng spiked into each sample) for quantification based on a full set of nine calibrators run with each set of samples. The calibration curve was weighted by the reciprocal of concentration (1/X) and typically resulted in correlation coefficients of >0.99. Samples with values below the lowest standard were designated as less than the lowest reportable value. Urine samples with values exceeding the highest standard were diluted into method linear range and reanalyzed. Instrument software (Analyst, MDS Sciex) generated a results spreadsheet that was imported into a relational database (Microsoft Access, Redmond, WA) using an automated, customwritten routine. Further data analysis, including quality control
Figure 1. Typical daily calibration curve for perchlorate based on analysis of nine standard solutions across the linear range. The values were weighted (1/X) by the Analyst software (PE Sciex) and produced excellent linear correlation coefficients (typically >0.99). The calibration curve spanned over 3 orders of magnitude with a working range from the lowest reportable value (0.025 ng/mL) to the highest standard (100 ng/mL).
(QC) evaluation, blank subtraction, and statistical analyses, was performed using SAS statistical software (SAS Institute, Cary, NC). Daily Operating Protocol. A typical daily sample batch included one reagent blank, 75 unknown samples, 2 high QC samples, 2 low QC samples, and a full set of 9 calibrators. A synthetic urine reagent blank was processed along with unknown urine samples to monitor for contamination. If that blank contained measurable amounts of perchlorate, then the entire data set was rejected and the source of the contamination removed before proceeding. Before daily instrumental analyses, a known standard was injected to confirm acceptable chromatographic resolution and mass spectral sensitivity. After instrumental verification, the blank was analyzed, followed by a full set of nine standards, two QCs, the unknowns, and the other two QC samples. All standards injected on that day were then used to generate a daily calibration curve with correlation coefficients typically greater than 0.99 (Figure 1). Daily preventative maintenance was required to maintain the system in good working order. In-line precolumn filters (0.5 µm) were changed daily to minimize the risk of line blockage or column fouling. Column bed support filters were changed if column back pressure increased above normal levels. The mass spectrometer curtain plate was cleaned daily to help minimize fouling of the mass spectrometer. We required both the reagent blank and QC materials to meet clear specifications before approving a batch of data. If a reagent blank exceeded the lowest reportable value, we rejected the batch and reprocessed the samples. A batch also could be rejected because of the values found for the QC materials. The criteria for rejection were a trend of 10 values above or below the mean, 1 value outside the upper or lower 3 standard deviation confidence interval, or 2 sequential values outside the 2 standard deviation confidence interval.26 Unknown samples analyzed simultaneously with QC material that produced unsatisfactory results were subsequently reprocessed and reanalyzed. All unknown samples (26) Westgard, J. O.; Barry, P. L.; Hunt, M. R.; Groth, T. Clin. Chem. 1981, 27, 493-501.
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Figure 2. Comparison of a human urine sample analyzed by both tandem mass spectrometry and conductivity. Panels A-C show MS/MS signal, and panel D shows conductivity trace for the same run. Panel A shows the perchlorate quantification ion transition of 99-83 m/z (100% relative intensity). Panel B shows the perchlorate confirmation ion transition of 101-85 m/z, and Panel C shows the 18O-labeled perchlorate internal standard ion transition 107-89 m/z. Panel D shows the conductivity signal measured in microsiemens. This urine sample contains 10 ng/mL perchlorate and 4 ng/mL internal standard.
were further evaluated for data quality to ensure proper signal from confirmation and internal standard ions. RESULTS AND DISCUSSION Unequivocal quantification of trace levels of perchlorate in human urine requires improved selectivity compared with existing methods. By coupling ion chromatography and tandem mass spectrometry, we achieved this objective by substantially reducing chemical noise. Comparison of a human urine sample analyzed using both tandem mass spectrometry and conductivity (Figure 2) demonstrates the magnitude of improved selectivity achieved for perchlorate quantitation in a difficult matrix. Note that the signal-to-noise ratio for the perchlorate quantitation ion trace (∼970) greatly exceeds the signal-to-noise ratio for the conductivity signal (∼19) that is composed of both the perchlorate analyte and internal standard. Clearly tandem mass spectrometry improves measurement of perchlorate in difficult matrixes, especially when coupled with ion chromatography. Reliable chromatography is crucial for the analysis of urinary perchlorate following minimal sample preparation. We found ion chromatography with the AS16 column to reliably resolve sulfate (2.0-min retention time) from perchlorate (6.0-min retention time). This resolution was consistent, with no degradation of chromatography or quantification following 75 repeated injections of first morning void urine. Reliable chromatography allowed early-eluting materials to be diverted to waste using a timed diversion valve. Other anions in the urine sample (e.g., phosphate) could partially elute with perchlorate but were resolved using the selectivity of tandem mass spectrometry. It is possible for coeluants to suppress ionization of perchlorate and 18O4-perchlorate internal standard. However, minimal ion suppression would not alter the ratio of perchlorate signal to internal standard signal and thus will not alter quantitation. 2478 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005
Excellent chromatographic resolution of perchlorate from potential interferences (e.g., sulfate) was accomplished using a Dionex AS16 column with an isocratic mobile phase of 50 mM sodium hydroxide. This nonvolatile mobile phase can rapidly foul the mass spectrometer interface. Volatile mobile phases, such as ammonium acetate, are more amenable to the mass spectrometer interface but are not sufficiently selective to elute the perchlorate from the column. Therefore, we chose a sodium hydroxide mobile phase followed by a postcolumn suppressor that efficiently removes the sodium ions before the eluant flows into the electrospray mass spectrometer interface. This analytical approach allowed us to use the most appropriate separation technique (ion chromatography) followed by the most selective detection (mass spectrometry) to enable detection of trace levels of perchlorate in human urine. The IC-MS MS method produced an excellent limit of detection (LOD), even in difficult matrixes such as urine. The LOD was calculated as 3S0, where S0 is the value of the standard deviation as the concentration approaches zero.27 S0 was determined by analyzing six sets of the lowest four calibration standards spiked into synthetic urine and plotting the standard deviation versus the known standard concentration. The y-intercept of the best-fit line of this plot was used as S0. The calculated values for 3S0 (0.003 ng/mL) and 10S0 (0.011 ng/mL) were lower than the lowest standard (0.025 ng/mL), and thus, the lowest reportable value for analysis of perchlorate in 0.5 mL urine was set at 0.025 ng/mL. Analysis of synthetic urine samples containing 0.025 ng/ mL perchlorate typically produces a quantitation ion peak with a signal-to-noise ratio exceeding 15:1. (27) Taylor, J. K. Quality Assurance of Chemical Measurements; Lewis Publishers: New York, 1987.
Perchlorate was quantified against calibrators ranging from 0.025 to 100 ng/mL. A broad linear range was required because of highly variable perchlorate levels in human urine specimens; preliminary human urine data indicate perchlorate levels vary by nearly 3 orders of magnitude between different individuals. Each batch of unknown samples was analyzed with a set of nine calibrators (one at each of nine different concentrations) and the relative response of those calibrators used to draw the calibration curve for that particular day. A typical calibration curve (weighted 1/X) is shown in Figure 1. The inset panel shows that the calibration curve has excellent linearity across the calibration range, including at lower concentrations (see inset plot of 0.0250.6 ng/mL). Repeated analysis of calibrators at each concentration level produced tight intraday results (0.025 ng/mL CV ) 10%; 100 ng/mL CV ) 3.1%), reinforcing the precision of this daily calibration procedure. Interday variability of daily calibration curve slopes was minimal; the relative standard deviation of 18 calibration curves analyzed over a four-week period was 4%. Despite the wide range of perchlorate concentrations measured in human samples (0.4-1100 ng/mL), we did not find problems with carryover signal from one sample to the next. We confirmed no measurable perchlorate contamination by lot screening all consumables and analyzing blanks with each batch of unknowns. For the method published here, no contamination problems were identified. However, we found perchlorate contamination during preliminary experiments with commercially available solid-phase extraction (SPE) cartridges containing ion exchange sorbents. SPE cartridges can selectively remove potential anionic interferences from salty aqueous samples,24 with chloride removed by silver sorbent, sulfate removed by barium sorbent, and phosphate removed by alumina.28 Urine is a challenging matrix for perchlorate measurement due to parts-perthousand levels of major anionic interferences (chloride, sulfate, phosphate); therefore, we evaluated ion exchange sorbents from two vendors. We found that ion exchange cartridges (silver and barium) from both vendors contained measurable levels of perchlorate (low-nanogram quantities per cartridge). Due to this contamination, the cost of SPE cartridges, and the amount of sample handling required, we chose to use minimal sample preparation coupled with a diversion valve that routes most of the urinary anions to waste before the perchlorate elutes. The combination of reliable ion chromatography and waste diversion produces a higher throughput method that is not affected by contamination in SPE cartridges. Due to the endogenous levels of perchlorate in human urine samples, we chose to prepare standards in synthetic urine. Perchlorate potentially could behave differently in actual human urine than it does in synthetic urine. Potential matrix effects on the calibration curve were evaluated by comparing standards prepared in synthetic urine with those prepared in human urine. Standards spiked into urine from a single volunteer produced a calibration curve with a substantial y-intercept due to endogenous perchlorate already in the sample (2.0 ng/mL); however, the slope was not significantly different from the slope produced by standards prepared in synthetic urine (slopes, 0.275 and 0.277, respectively). Therefore, no interfering matrix effect was observed (28) Ellington, J. J.; Wolfe, N. L.; Garrison, A. W.; Evans, J. J.; Avants, J. K.; Teng, Q. Environ. Sci. Technol. 2001, 35, 3213-8.
Table 1. Method Accuracy and Precision sample
N
proficiency test A proficiency test B proficiency test C proficiency test D QC lowb QC high unspiked urine spiked 1 ng/ml spiked 5 ng/ml spiked 50 ng/ml
5 5 5 7 20 20 4 4 4 4
a
theoretical (ng/mL)
mean (ng/mL)
std dev
CV, %a
% diff
0.19 2.4 12 72 5.0 75
0.19 2.29 11.42 69.14 4.92 75.09 4.34 5.29 9.31 54.50
0.01 0.08 0.55 2.13 0.21 2.08 0.03 0.03 0.02 0.20
7.6 3.3 4.8 3.1 4.2 2.8 0.7 0.7 0.2 0.4
2.1 4.4 4.8 3.8 1.6 -0.1
5.3 9.3 54.3
5.0 0.4 -0.3
Coefficient of variation. b Quality control.
for the range of analyte concentrations measured (0.2-100 ng/ mL), and calibration curves were subsequently produced using data collected by analyzing standards prepared in synthetic urine. The potential for variable urine components to interfere with perchlorate quantification was greatly reduced by the use of 18O4perchlorate as an internal standard. Suppression or enhancement of ionization can alter absolute signal for perchlorate and 18O4perchlorate, but the ratio of analyte to internal standard will remain constant. Method accuracy was evaluated using three different perchlorate-containing media: proficiency testing material, QC material, and perchlorate-spiked human urine. Proficiency testing specimens were prepared at four concentrations by diluting certified reference solutions of ammonium perchlorate with synthetic urine. The resulting proficiency testing samples were blind analyzed and reported to an external QC officer for evaluation (data in Table 1). Note that the measured amounts deviate minimally (2-5%) from the true amount. Method accuracy was also calculated when QC pools were prepared by spiking known amounts of perchlorate into perchlorate-free synthetic urine. These QC pools were then analyzed 20 times over a 10-day period and the mean characterized values compared with the amounts of perchlorate spiked into each sample. The measured amounts of perchlorate deviated minimally (-0.1 to 1.6%) from the amount expected based on the spiked amounts (Table 1). Finally, method accuracy was also evaluated by spiking perchlorate into human urine and quantifying the amount of perchlorate recovered. This method was complicated by the fact that the human urine used for this experiment already contained measurable amounts of perchlorate (4.3 ng/mL); once endogenous perchlorate levels were included in the calculations, the method was shown to be similarly accurate in human urine as it had proven to be with synthetic urine experiments, with differences ranging from -0.3 to 5.0%. The proficiency testing and quality control materials were also used to evaluate assay precision. The single-day experiments involving proficiency testing materials and spiked matrix showed excellent precision (all CVs 5.45 or 100-fold) within each city population. This variability likely resulted from varied water use and dietary habits.31,32 Our results from both the Atlanta and Chile study populations are consistent with other published measurements of urinary perchlorate. Previous methods for measuring perchlorate have not been adequately sensitive for quantifying perchlorate in urine samples from unexposed populations; therefore, application of these methods has resulted in reported background values of less than methodological LODs of 500,33 20,9,34 and 5 ng/mL.35,36 Gibbs et al.36 recently measured perchlorate levels in 30 urine samples collected from children in Taltal, Chile, in 1999-2000:36 urinary perchlorate ranged from 25 to 880 µg/g of creatinine, with a median value of 180 µg/g of creatinine. These data compare favorably with our analysis of urine samples collected from women living in Taltal and drinking the same perchlorate-contaminated (29) Exploratory Data on Perchlorate in Food; Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Plant and Dairy Foods. 2004. http://www.cfsan.fda.gov/∼dms/clo4data.html. (30) Fresh Fruit and Vegetable Shipments; United States Department of Agriculture, 2003. http://www.ams.usda.gov/fv/mncs/shipsumm03.PDF (31) Te´llez, R.; Michaud, P.; Reyes, C.; Blount, B. C.; Vanlandingham, C. B.; Crump, K. S.; Gibbs, J. P. Thyroid. In press. (32) Orris, G. J.; Harvey, G. J.; Tsui, D. T.; Eldrige, J. E. Preliminary analyses for perchlorate in selected natural materials and their derivative products. OpenFile Report 03-314, 1-6. 2002. http://geopubs.wr.usgs.gov/open-file/of03314/OF03-314.pdf (33) Lawrence, J. E.; Lamm, S. H.; Pino, S.; Richman, K.; Braverman, L. E. Thyroid 2000, 10, 659-63. (34) Merrill, E. A.; Clewell, R. A.; Robinson, P. J.; Jarabek, A. M.; Gearhart, J. M.; Sterner, T. R.; Fisher, J. W. P. Toxicol. Sci. 2004, 83 (1), 25-43. (35) Crump, C.; Michaud, P.; Tellez, R.; Reyes, C.; Gonzalez, G.; Montgomery, E. L.; Crump, K. S.; Lobo, G.; Becerra, C.; Gibbs, J. P. J. Occup. Environ. Med. 2000, 42, 603-12. (36) Gibbs, J. P.; Narayanan, L.; Mattie, D. R. J. Occup. Environ. Med. 2004, 46, 516-7.
tap water: urinary perchlorate ranged from 15 to 700 µg/g of creatinine, with a median value of 119 µg/g of creatinine. Urinary perchlorate is an effective biomarker for perchlorate exposure. Not only is urine collection less invasive than blood collection, but urinary perchlorate levels tend to be much higher than serum levels due to efficient renal clearance of perchlorate. These advantages will allow us to assess perchlorate exposure in large populations (e.g., National Health and Nutrition Examination Survey, NHANES) and report the data as part of the National Report on Human Exposure to Environmental Chemicals.37 We plan to develop related IC-MSMS methods for quantifying perchlorate in other human matrixes. Serum is of special interest because it is the biological fluid that delivers perchlorate to the target tissue (thyroid). The finding of perchlorate (even at trace levels) in all human urine samples tested indicates the likelihood of widespread tracelevel perchlorate exposure in the general population. We are currently evaluating this possibility by quantifying perchlorate in urine samples collected from a large national exposure study (NHANES). Further research is needed to characterize general population exposure to perchlorate and the sources of this exposure. Although our method is effective for quantifying environmental perchlorate exposure, it does not distinguish between synthetic and natural perchlorate exposure sources. Bao and Gu38 recently published provocative methods for differentiating synthetic and natural perchlorate by quantifying subtle differences in oxygen and chlorine isotope ratios. Their method is not applicable to largescale biomonitoring applications due to inadequate throughput and sensitivity but offers a complimentary technique for exposure source identification. Our initial measurements indicate that perchlorate exposure is widespread, albeit at trace levels. The toxicological impact of chronic trace-level perchlorate exposure is uncertain. The National Research Council (NRC) recently defined a reference dose for (37) National Report on Human Exposure to Environmental Chemicals; Centers for Disease Control and Prevention, 2004. http://www.cdc.gov/exposurereport/ (38) Bao, H.; Gu, B. Environ. Sci. Technol. 2004, 38 (19), 5073-5077. (39) Tonacchera, M.; Pinchera, A.; Dimida, A.; Ferrarini, E.; Agretti, P.; Vitti, P.; Santini, F.; Crump, K.; Gibbs, J. Thyroid 2004, Dec. 14 (12), 1012-9. (40) National Research Council, Health Implications of Perchlorate Ingestion; The National Academies Press: Washington, 2005.
perchlorate at 0.0007 mg/kg‚day.40 Only one sample from the Atlanta convenience population contained perchlorate at levels in excess of the amount expected to be excreted by an individual exposed to perchlorate at the NRC reference dose. The potential health effects of trace-level perchlorate exposure will need to be addressed in future studies that combine perchlorate biomonitoring with assessment of thyroid function. Additional perspective on perchlorate exposure is gained by examining exposure to other substances that compete with iodide for absorption into the thyroid gland.39 Americans typically are exposed to significant quantities of other antithyroid agents (e.g., nitrate and thiocyanate) that can also inhibit iodide uptake by the thyroid. Nitrates occur naturally in many vegetables and are found in water and preserved meats. Thiocyanates are found in tobacco smoke, milk, and some vegetables. Compared with perchlorate, nitrate and thiocyanate have lower affinities for the sodium iodide symporter, but serum levels of these anions tend to be much higher. Thus, the thyroid-inhibiting potential of a given perchlorate dose must be compared with the potential inhibitory effects of nitrate and thiocyanate to which the thyroid also may be exposed. Further research is needed to better understand the relative health significance of perchlorate exposure. We have developed and validated an improved method for assessing perchlorate exposure by quantifying perchlorate in human urine. This rapid, selective, and sensitive method will help to elucidate the human health relevance of environmental perchlorate exposure. ACKNOWLEDGMENT The use of trade names is for identification purposes only and does not constitute endorsement by the U.S. Department of Health and Human Services or the Centers for Disease Control and Prevention. All work on human samples was approved by the CDC Institutional Review Board (CDC protocols 4316 and 3465). The authors thank Dr. David Ashley for valuable suggestions, John Morrow for data assistance, and Dr. John Gibbs for the use of residual urine samples from the Chilean study: “Chronic Environmental Exposure to Perchlorate and Thyroid Function During Pregnancy and the Neonatal Period”. Received for review November 4, 2004. Accepted January 26, 2005. AC048365F
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