Environ. Sci. Technol. 2008, 42, 2522–2527
Method for Quantifying Nitromethane in Blood as a Potential Biomarker of Halonitromethane Exposure K . U D E N I A L W I S , * ,‡ BENJAMIN C. BLOUNT,‡ LALITH K. SILVA,‡ MITCHELL M. SMITH,‡ AND KARL-HERMANN LOOSE§ Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, and Thermo Fisher Scientific, Bremen, Germany
Received October 30, 2007. Revised manuscript received December 13, 2007. Accepted January 2, 2008.
The cytotoxicity and genotoxicity of nitromethane and its halogenated analogues in mammals raise concerns about potential toxicity to humans. This study shows that halonitromethanes are not stable in human blood and undergo dehalogenation to form nitromethane. We quantified nitromethane in human blood using solid-phase microextraction (SPME) headspace sampling coupled with gas chromatography (GC) and high resolution mass spectrometry (HRMS). The limit of detection was 0.01 µg/L with a linear calibration curve spanning 3 orders of magnitude. This method employs isotope dilution to precisely quantify trace amounts of nitromethane (coefficient of variation 98% purity) were purchased from Aldrich Chemicals Co. (St. Louis, MO). Chloronitromethane, dichloronitromethane, dibromoni10.1021/es702733k CCC: $40.75
2008 American Chemical Society
Published on Web 02/23/2008
TABLE 1. Spike Recovery Data from Blood Collected from Three Healthy Volunteers and Spiked at Three Different Levels of Nitromethanea
Level I (n ) 9) Level II (n ) 9) Level III (n ) 9)
mean spiked amount µg/L
mean difference (total - unspiked blood)b µg/L
mean spike recoveryb %
1.01 2.08 3.08
0.89 ( 0.20 2.03 ( 0.15 3.05 ( 0.27
88 ( 17 98 ( 8 99 ( 5
a
Mean unspiked blood levels of volunteers were 0.63, 0.93, and 1.28 µg/L. b Average ( standard deviation.
tromethane, and tribromonitromethane (bromopicrin) were purchased from (Orchid Helix, Toronto, Canada). 13C-labeled nitromethane was purchased from Cambridge Isotopes (Andover, MA). The purity levels of nitromethane (99%) and the labeled analogue (90%) were determined using total-ion chromatograms constructed from full-scan mass data and liquid injections in methanol. The analytes were stored at 4 °C. The labeled internal standard was stored at -20 °C to minimize degradation. Blood Sample Collection. Blood samples for VOC analysis were collected in 10-mL blood-collection vials (Vacutainers, Becton-Dickinson, Franklin Lakes, NJ) that had been treated with heat and vacuum to minimize volatile organic contaminants (15). Blood samples were collected by venipuncture (16). Immediately after the collection, each blood sample was mixed thoroughly on a blood mixer or manually to dissolve the anticoagulants (sodium fluoride/potassium oxalate) and stored at 4 °C in the dark until analysis. Individual consent of study participants and the human subjects (IRB) approval were obtained prior to this study. The anonymous subjects were multiethnic, both male and female, and 12–86 yr of age. Blood Sample Analysis Using SPME. Prior to analysis, blood samples were allowed to equilibrate to room temperature while being mixed with a hemo-mixer for approximately 20 min. After mixing, 3 mL of blood was removed from each vacutainer using a precleaned 5-mL glass-barrel gastight syringe (Hamilton, Reno, NV) and transferred into a tared 10-mL SPME vial. Internal standard solution (18.4 pg 13C labeled nitromethane in 40 µL) was added to the vial, which was crimp-sealed immediately with Teflon-lined silicone septum and steel/aluminum crimp seal and then reweighed. Blanks, standards, and quality control (QC) samples were prepared similarly. The plungers and barrels of the sampling syringes, which were prebaked overnight at 80 °C in a vacuum oven, were rinsed with purge-and-trap grade methanol and rinsed twice with VOC-free water immediately before use. The SPME vials and septa were heated at 80 °C in a vacuum oven until use. Spike Recovery. Blood samples from three healthy volunteers were used for the spike recovery assay. Approximately 40 mL of blood of each subject was transferred from four vacutainers into separate serum bottles (60 mL capacity) and mixed gently for 5 min. After mixing, approximately 13 mL blood from each serum bottle was transferred into three small (20 mL capacity) serum bottles. Blood (3 mL) from each small serum bottle was transferred to SPME vials to determine the background level of nitromethane in the blood. The remaining blood (∼10 mL) in the three small serum bottles for each subject was spiked separately with three different levels of nitromethane (Table 1) and mixed on a blood mixer for 20 min. After mixing, three aliquots (3 mL each) of each spike level were transferred to nine SPME vials for each volunteer. Internal standard (40 µl) was added to both spiked and unspiked blood samples in SPME vials, crimp sealed, and queued in a Peltier cooled
rack (15 ( 1 °C) for SPME/GCHRMS analysis. Blank blood and spiked blood samples were assayed in triplicate for each subject.
Time-Course Experiments with Trichloronitromethane and Tribromonitromethane Spiked Blood The hydrolysis of trichloronitromethane and tribromonitromethane was studied by spiking these compounds into blood samples and blood plasma. Trichloronitromethane and tribromonitromethane standard solutions were prepared in methanol and stored at -20 °C. Blood (3 mL) in a SPME vial was spiked with 20 µg/L trichloronitromethane and then with internal standard (40 µL). Blank blood sample (3 mL blood plus 40 µL internal standard) was prepared in the same manner. The spiked blood and the blank blood samples were run immediately after preparation, every 4 h up to 24 h, and then at 48 h, 96 h, and after a week. Similarly, tribromonitromethane-spiked (37 µg/L) blood and a blank blood sample were prepared and run at the same time intervals. Blood Spike Experiments with Peroxynitrite. Peroxynitrite-mediate formation of nitromethane was studied by spiking peroxynitrite into blood samples. Sodium peroxynitrite was purchased from Cayman Chemicals (34.4 mM in 0.3 M NaOH, Ann Arbour, MI) and stored at -70 °C until analysis. Blood samples (3 mL) were spiked separately with 20, 30, 40, 60 µL of sodium peroxynitrite, and then with internal standard (40 µL), and analyzed for nitromethane concentrations immediately after preparation. To check whether peroxynitrite was contaminated with nitromethane, peroxynitrite (20 µL) in phosphate buffer (3 mL, pH 7.4) was spiked with internal standard (40 µL) and analyzed for nitromethane. A blank buffer sample was also prepared similarly but without adding peroxynitrite for comparison. SPME/GCHRMS Analysis. The solid-phase microextraction coupled with GC-HRMS method for halonitromethanes was developed on a MAT 95XP magnetic sector MS (Thermo Fisher Scientific Inc., Waltham, MA) combined with a 6890 Agilent GC (Palo Alto, CA) and a CombiPAL autosampler (CTC Analytics AG, Zwingen, Switzerland). Helium (research grade 99.9999%, Airgas, Atlanta, GA) was used as the carrier gas with a constant flow of 0.7 mL/min. The GC was held at -1 °C for 3 min initially and then increased to 35 °C at a rate of 50 °C/min with a holding time of 1 min followed by a rate of 10 °C/min to 140 °C and finally ramped at 50 °C/min to 220 °C, which was held for 3 min. The transferline was kept at 220 °C. More details about the SPME and the GC method can be found in the Supporting Information. The high resolution mass spectrometer was operated in EI positive ion mode with multiple ion detection at 10000 resolution. The source was kept at 240 °C and the reference inlet at 150 °C. Perfluorokerosene (m/z 68.9947, lock mass) and n-butyl benzene (m/z 91.0542 calibration mass) were used as mass calibrants (17). We monitored CH3NO2+ · (m/z 61.0158, quantitation ion), CH2NO2+ · (m/z 60.0080, confirmation ion) for the native, and 13CH3NO2+ · (m/z 62.0192) for the labeled analogue. The dwell time for lock and calibration masses were 7 msec and for the analytes were 142 msec, the cycle time was 0.5 s. For the biomarker study, chloronitromethane (CNM), dichloronitromethane (DCNM), trichloromethane (TCNM), and their brominated analogues were determined simultaneously, but not quantified, with nitromethane. Quantification. Xcalibur software (version 1.3, Thermo Finnigan, Breman, Germany) was used for peak integration, calibration, and quantification. Relative response factors were calculated on the basis of the ratio of relative peak area of the analyte quantification ion to that of the labeled analogue ion. A set of eight calibration standards was analyzed with each set of unknown samples. A weighted, 1/x (where x is VOL. 42, NO. 7, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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the standard concentration), least-squares model was fit to the calibration and typically resulted in linear curves with r2 >0.999. Ion ratios were adjusted for cross-contamination between native analyte and labeled analogue according to Colby and McCaman (18). The limit of detection (3S0) was determined by analyzing six sets of calibration standards. The calculated value of 3S0 was lower than the lowest standard, therefore, the lowest standard was used as the lowest reportable concentration (19). Each analytical run consisted of a set of calibration standards, two QC samples (low-concentration QC and high-concentration QC), the blank water, and unknowns. Quality Assurance. Labeled analogue response was evaluated on the basis of absolute peak area signal as well as signal-to-noise ratio as an instrument response check. We further evaluated the identity of the analyte ion by comparing GC retention time and the confirmation-toquantitation ion ratio in unknown samples with that of reference standards. A full scan of a blood sample obtained at 2000 resolution (mass range 29 to 70 amu) and a NIST library match confirmed the presence of nitromethane in human blood. A blank water sample was included in each analytical batch to test for any contamination. Blank water was prepared by helium sparging, distilling, and flame sealing in glass ampoules. Additionally, a SPME fiber sampling of laboratory air was run to assess airborne contaminants qualitatively. Quality Control Samples. Two low-concentration QC (QCL) and two high-concentration QC (QCH) samples were included in each analytical batch. Bovine serum (Hyclone Laboratories, Logan, UT) was used for QC preparation. A high QC pool was prepared by spiking a known amount of a concentrated standard into bovine serum. For the low QC pool, we used bovine serum without spiking because unspiked bovine serum contained a low level of nitromethane. Subsequently, aliquots were stored at -70 °C in crimp-sealed serum bottles (glass,10 mL capacity). Proficiency Testing. To prepare proficiency testing (PT) material we purchased nitromethane (>99% purity) from Acros Chemicals (Morris Planes, NJ). Four concentrations of PT standards were prepared in methanol. Samples of each of these concentrations was transferred into vacules (1 mL) and flame sealed. These vacules then were blind-coded by an independent QC officer and stored at -70 °C. PT samples were analyzed every six months or after major instrument maintenance. The assay passed PT if blind-analyzed amounts fell within 25% of the actual values.
Results and Discussion We developed a sensitive, accurate, and precise analytical method to detect nitromethane in human blood. This biomonitoring method can be used for exposure assessment to determine potentially toxic halonitromethane disinfection byproducts, workers exposed to nitromethane or halonitromethanes or both in occupational environments, and to determine chloropicrin exposure in chemical terrorism investigations. The GC conditions and the SPME extraction temperature were optimized for nitromethane analysis. The injector port and the transferline temperature were kept at 170 and 200 °C, respectively, to prevent thermal decomposition of trihalonitromethanes (3). We investigated the effect of SPME extraction temperature on blood samples by extracting samples at different SPME temperatures (30–60 °C). The variability of blood nitromethane concentrations obtained at different SPME extraction temperatures was minimal (relative standard deviation [RSD] ) 5.7%). However the SPME extraction temperature was set to 30 °C because the absolute responses obtained for both the analyte and the 2524
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FIGURE 1. Typical chromatogram resulting from the analysis of nitromethane in human blood (0.55 µg/L).
FIGURE 2. Calibration curves for nitromethane in water and blood. labeled analogue were higher at 30 °C than those obtained at other temperatures tested. Figure 1 shows a typical chromatogram that indicates the results of the analysis of a human blood sample for nitromethane. The quantitation (m/z 61.0158), confirmation (m/z 60.008), and the 13C- labeled analogue (m/z 62.0192) ions were well resolved from potentially interfering compounds in blood samples. The clean ion chromatogram was the results of mass analysis at 10000 resolution (5% valley definition), chromatographic resolution, and VOC extraction from the headspace above blood level. For this method water- instead of serum- or blood-based calibration standards were used because we detected a wide variation in the background nitromethane concentrations in blood samples from people with no known exposure. The water-based calibration was linear throughout the range (0.01–12 µg/L). A comparison between water- and blood-based calibration (Figure 2) showed that the slope of both curves was almost the same, suggesting minimal matrix effect between the two matrices for nitromethane analysis. However, the blood-based calibration had a larger intercept due to background nitromethane concentrations found in blood. Both calibrations were linear (r2 >0.999) and spanned 3 orders of magnitude. The limit of detection for this method was 0.01 µg/L. Vacutainer stoppers were checked for nitromethane contamination. Nitromethane was not detected in VOC-free,
water-filled (10 mL) vacutainers stored (some vertically and some horizontally) at 4 °C for 6 days. To further confirm this finding, blood samples from three volunteers were drawn directly to separate blood syringes (without collecting into vacutainers) which had been precleaned with methanol and VOC-free water and assayed for nitromethane immediately. The nitromethane concentrations determined in these samples were the same as those determined in blood collected into vacutainers from these same volunteers. The short-term stability of the analyte was studied for prepared blood samples (3 mL blood in SPME vials spiked with 40 µL internal standard). The analyte was stable for 48 h at 15 °C with a RSD
