Anal. Chem. 1999, 71, 3526-3530
Isotope Dilution High-Performance Liquid Chromatography/Tandem Mass Spectrometry Method for Quantifying Urinary Metabolites of Atrazine, Malathion, and 2,4-Dichlorophenoxyacetic Acid M. D. Beeson,* W. J. Driskell, and D. B. Barr
National Center for Environmental Health, Centers for Disease Control and Prevention (CDC), 4770 Buford Highway, NE (F-17), Atlanta, Georgia 30341
We have developed an isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/ MS/MS) method for quantifying the urinary metabolites of the pesticides atrazine, malathion, and 2,4-dichlorophenoxyacetic acid (2,4-D). Urine samples are extracted with an organic solvent, and the organic fraction is concentrated. The concentrate is then analyzed using HPLC/MS/MS. The limits of detection for the metabolites are less than 0.5 µg/L (parts per billion) in 10 mL of urine, with a high degree of accuracy and precision. Currently, there is much concern about the exposure of humans to pesticides.1-3 Although most people are not occupationally exposed to pesticides, nearly everyone has some level of exposure resulting from food, air, water, or dermal contact. Some of the most widely used pesticides include the herbicides atrazine and 2,4-dichlorophenoxyacetic acid (2,4-D), usually applied as the salt of 2,4-D or as the 2,4-D ester, and the insecticide malathion.4 Many studies have been conducted to determine the possibility of a causal relationship between these pesticides and health outcomes, such as non-Hodgkin’s lymphoma, soft tissue sarcoma, breast cancer, and endocrine disruption effects.5-7 * Corresponding author: (e-mail)
[email protected]; (fax) 770-488-7609. (1) Hoar Zahm, S.; Ward, M. H. Environ. Health. Perspect. 1998, 106 (Suppl. 3), 893-908. (2) Landrigan, P. J.; Carlson, J. E.; Bearer, C. F.; Spyker Cranmer, J.; Bullard, R. D.; Etzel, R. A.; Groopman, J.; McLachlan, J. A.; Perera, F. P.; Reigart, J. R.; Robison, L.; Schell, L.; Suk, W. A. Environ. Health. Perspect. 1998, 106 (Suppl. 3), 787-794. (3) Hill, R. H., Jr.; Head, S. L.; Baker, S.; Gregg, M.; Shealy, D. B.; Bailey, S. L.; Williams, C. C.; Sampson, E. J.; Needham, L. L. Environ. Res. 1995, 71, 99-108. (4) Aspelin, A. L. Pesticide Industry Sales and Usage; U.S. Environmental Protection Agency. Office of Prevention, Pesticides and Toxic Substances. U.S. Government Printing Office: Washington, DC, 1997; EPA-733/R-97/ 002. (5) Risk Assessment Forum. Special Report on Environmental Endocrine Disruption: An Effects Assessment And Analysis; U.S. Environmental Protection Agency. U.S. Government Printing Office: Washington, DC, 1997; EPA630/R-96/012. (6) Aprea, C.; Sciarra, G.; Bozzi, N. J. Anal. Toxicol. 1997, 21, 262-267. (7) Kettles, M. A.; Browning, S. R.; Prince, T. S.; Horstman, S. W. Environ. Health Perspect. 1997 105, 1222-1227.
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Because of the widespread use of these pesticides, a strong need exists for accurate biomonitoring methods to assess exposure of these pesticides. Nonmetabolized pesticides can be analyzed in serum; however, in many public health studies, urine samples are more easily obtained than blood samples. In urine, more pesticides are present in their metabolized form; thus urinary biomonitoring for a pesticide requires knowledge of its metabolite(s). Atrazine mercapturate was identified as a primary metabolite of atrazine,8 and a previous study in which the National Center for Environmental Health analyzed the urine of farm families exposed to atrazine confirmed this finding.9 The pesticide 2,4-D is primarily excreted in urine unchanged, and 2,4-D esters are hydrolyzed in vivo and excreted as 2,4-D.10,11 The major metabolites of malathion are the carboxylic acid of malathion,12 the recovery of which has been reported to be variable,13 and a dicarboxylic acid, which generally presents no extraction problems.14 At low levels of exposure, the diacid is observed in greater abundance.12 Current methods of analysis for the malathion diacid and 2,4-D metabolites typically include derivatization followed by gas chromatography with electron capture detection (GC/ECD), GC with a nitrogen/phosphorus detector (GC/NPD), or GC with mass spectrometric detection (GC/MS). The derivatization is often timeconsuming, in some cases requiring an overnight reaction followed by an additional cleanup before analysis.6,12-16 Analysis methods for atrazine metabolites include liquid scintillation counting, (8) Lucas, A. D.; Jones, A. D.; Goodrow, M. H.; Saiz, S. G.; Blewett, C.; Seiber, J. N.; Hammock, B. D. Chem. Res. Toxicol. 1993, 6, 107-116. (9) Hill, R. H.; Driskell, W. J.; Head, S. L.; Needham, L. L.; Bond, A. E., CDC, unpublished results. (10) Sauerhoff, M. W.; Braun, W. H.; Blau, G. E.; Gehring, P. J. Toxicology 1977, 8, 3-11. (11) Knopp, D.; Glass, S. Int. Arch. Occup. Environ. Health 1991, 63, 329-333. (12) Bradway, D. E.; Shafik, T. M. J. Agric. Food Chem. 1977, 25, 1342-1344. (13) Draper, W. M.; Wijekoon, D.; Stephens R. D. J. Agric. Food Chem. 1991, 39, 1796-1801. (14) Fenske, R. A. In Biological Monitoring for Pesticide Exposure; Wang, R. G. M., Franklin, C. A., Honeycutt, R. C., Reinert, J. C., Eds.; ACS Symposium Series 382; American Chemical Society: Washington, DC, 1989; pp 70-84. (15) Kutz, F. W.; Cook, B. T.; Carter-Pokras, O. D.; Brody, D.; Murphy, R. S. J. Toxicol. Environ. Health 1992, 37, 277-291. (16) Hill, R. H., Jr.; Shealy, D. B.; Head, S. L.; Williams, C. C.; Bailey, S. L.; Gregg, M.; Baker, S. E.; Needham, L. L. J. Anal. Toxicol. 1995, 19, 323-329. 10.1021/ac990130u CCC: $18.00
© 1999 American Chemical Society Published on Web 07/14/1999
accelerator mass spectrometry, high-pressure liquid chromatography with UV detection (HPLC/UV), and GC/NPD.17,18 Liquid scintillation counting requires the use of radioactive isotopes, while accelerator mass spectrometry is not accessible to most laboratories. HPLC/UV is accessible to many laboratories, but does not have the specificity available from high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Additionally, most analysis methods do not simultaneously analyze atrazine mercapturate, malathion diacid, and 2,4-D in a single method, although these are metabolites from some of the most widely used pesticides.4 In this paper we present a single method for quantifying the pesticide metabolites atrazine mercapturate, malathion dicarboxylic acid, and 2,4-D in human urine. Although the chemical diversity of the analytes requires the use of general sample preparation and HPLC conditions, it is logical to combine the analysis of these analytes into a single method. The sample preparation is simple and does not involve derivatization; the method is highly specific and yields results that are precise and accurate. EXPERIMENTAL SECTION Because of the possibility of being exposed to various microbiological hazards through handling the urine samples, direct contact with the specimen should be avoided. A Hepatitis B vaccination series is usually recommended for health care and laboratory workers who are exposed to human fluids and tissues. All solvents were analytical grade and were obtained from Burdick & Jackson Co. (Muskegon, MI), B&J label. Atrazine mercapturate was a generous gift from Dr. Bruce Hammock at the University of California (Davis). Malathion dicarboxylic acid was obtained from the U.S. Environmental Protection Agency Pesticides Repository (Research Triangle Park, NC). The 2,4dichlorophenoxyacetic acid was purchased from Sigma Chemical Co. (St. Louis, MO). D7-Labeled malathion dicarboxylic acid was provided by the State of California Department of Health Services. 13C -Labeled atrazine mercapturate and 13C -labeled 2,4-D were 6 6 purchased from Cambridge Isotope Labs, Inc. (Andover, MA). The gases used by the mass spectrometer were obtained from Holox (Norcross, GA) or Air Products and Chemicals, Inc. (Atlanta, GA) and had a minimum purity of 99.999%. All chemicals, solvents, and gases were used as obtained, with no further purification. Standard Preparation and Characterization. (a) Native Standards. Stock solutions of each analyte of approximately 200 ng/µL were made by dissolving each analyte in acetonitrile. An aliquot of each stock solution was combined to make seven solutions of varying concentrations. Concentrations were calculated such that a 100-µL aliquot of working standard in 10 mL of urine produced the desired standard concentration (ranging from 0.5 to 50.0 µg/L). The range of standards was designed to encompass the range of metabolite concentrations observed in the general population, which most probably result from incidental, not occupational, exposure. (17) Gilman, S. D.; Gee, S. J.; Hammock, B. D.; Vogel, J. S.; Haack, K.; Buchholz, B. A.; Freeman, S. P. H. T.; Wester, R. C.; Hui, X.; Maibach, H. I. Anal. Chem. 1998, 70, 3463-3469. (18) Ikonen, R.; Kangas, J.; Savolainen, H. Toxicol. Lett. 1988, 44, 109-112.
(b) Labeled Standards. Stock solutions of each labeled isotope of approximately 200 ng/µL were made by dissolving the labeled isotopes in acetonitrile. A 250-µL sample of each of the labeled stock isotopes was aliquotted into 5 mL of acetonitrile and mixed. The concentration of labeled isotope was designed to be close to the expected measured values of the unknown samples as determined in preliminary experiments. The combined labeled isotope solution was the internal standard (ISTD). Quality Control Materials. The quality control (QC) materials consisted of two urine pools collected from volunteers and enriched with known amounts of pesticide residues. Each pool was screened to ensure that the endogenous levels of pesticide residues were low or nondetectable. The pools were combined and homogenized to form a base pool. The base pool was split equally into three subpools. One subpool was reserved for blank and standard analyses (see Sample Preparation section). Another of the subpools was enriched with an appropriate amount of the stock solution of each pesticide residue to yield an approximate concentration of 8 µg/L (low pool). The final subpool was enriched with an appropriate amount of each pesticide stock solution to yield an approximate concentration of 20 µg/L (high pool). All QC pools were mixed thoroughly for 24 h following enrichment. Each pool was clean filtered to 0.2 µm. All QC pools were characterized to determine the mean and 99th and 95th control limits by consecutively analyzing at least 20 samples from each QC pool. Sample Preparation. Urine (10 mL) in 50-mL screw-capped test tubes was enriched with 100 µL of the ISTD. The pH of the urine was adjusted to approximately 3.7, as measured by pH paper, by adding 10% sulfuric acid. A 7.5-mL aliquot of a solution of 1 part methylene chloride-4 parts ethyl ether were added to each pH-adjusted sample. The sample was placed on a sample rotator (Glas-Col Apparatus Co., Terre Haute, IN) to ensure thorough mixing, for 5 min at 30 rpm and then centrifuged for 10 min at 2000 rpm. The organic layer was transferred to a clean 15-mL conical bottom tube. The extraction step was repeated, including rotation and centrifugation. The organic layer was transferred to the tube containing the previous extractant, and the combined extractants were concentrated to approximately 50 µL using a TurboVap LV evaporator (Zymark Corp., Framingham, MA) set at 50 °C. Acetonitrile (100 µL) was added to the concentrated extractant and the sample was then transferred to a vial suitable for automated injection onto the HPLC. The sample injection volume was 20 µL. Instrumental Analysis. The HPLC/MS/MS analyses were performed on a Finnigan TSQ-7000 triple quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an atmospheric pressure chemical ionization interface (APCI), which in turn was interfaced to a Waters Alliance 2690 HPLC (Waters Corp. Milford, MA). The analytes were chromatographically separated using a Whatman Partisil 5 ODS-3 column (4.6 mm × 25 cm) at a flow rate of 1.0 mL/min under isocratic conditions. The mobile phase was 60% (v/v) acetonitrile-40% water with 0.2% (v/v) glacial acetic acid. Extract (20 µL) was injected using the Waters Alliance autosampler. Two injections were made from each vial: one for detection of the 2,4-D and malathion diacid in negative ion mode and one for detection of the atrazine mercapturate in positive ion mode. Analytical Chemistry, Vol. 71, No. 16, August 15, 1999
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Table 1. Analytea Masses
Table 2. Analyte Detection Limits analyte
S0a
3S0
ATZ MDA 2,4-D
0.18 0.16 0.14
0.53 0.49 0.43
a S represents the intercept of a linear regression line fit to the 0 data points of the individual calibration curves and is in units of µg/L (ppb). See text. ATZ, atrazine mercapturate; MDA, malathion diacid.
Table 3. Recoveries of Pesticide Metabolites at Two Concentrations recovery (%) analyte
5 µg/La
10 µg/La
ATZ MDA 2,4-D
27 ( 6 75 ( 6 59 ( 11
14 ( 26 65 ( 8 64 ( 18
a Final concentration in prepared sample. ATZ, atrazine mercapturate; MDA, malathion diacid.
a
ATZ, atrazine mercapturate; MDA, malathion diacid.
Mass analyses consisted of daughter ion scans using the mass decompositions listed in Table 1. For quantification, one decomposition was monitored for each analyte and its analogous internal standard. When available, a second decomposition was monitored for confirmation. The corona of the APCI was 5 µA, and the heated capillary temperature was 250 °C. Argon was used as the collision gas for the collision-induced dissociation (CID). The pressure in the collision cell was approximately 2 mT and the collision energy was (22.0 eV, as appropriate. The electron multiplier was set at 2200 V. The analysis conditions were recorded into an acquisition program that was initiated upon injection by the HPLC autosampler. For analysis of the malathion diacid and 2,4-D, the total run time was 7 min. The atrazine mercapturate analysis time was 9 min. Data Processing and Analysis. Data were processed automatically by the analysis software provided with the mass spectrometer. The retention times of the ions of interest were determined, and the areas of the peaks corresponding to the ions of interest were calculated. The data were checked by the analyst for appropriate peak selection, baseline estimation, and interferences, which were corrected if necessary. Interferences were easily identified by the change in the ratio of quantification to confirmation ion, when the analysis permitted, as in the case of 2,4-D. After the data were transferred to a PC, statistical analyses were performed using SAS statistical software (SAS Institute, Inc. Cary, NC). Quantification. The samples, QC materials, standards, and blanks were all treated identically. The calibration curve was determined from standard solutions spiked into blank urine and extracted. The calibration curve consisted of at least five replicates of seven different analyte concentrations plotted against the response factors of the various analytes. The lowest standard was near the limit of detection (LOD) to ensure a linear calibration 3528 Analytical Chemistry, Vol. 71, No. 16, August 15, 1999
curve in this area. The response factors were calculated using published methods.19,20 The concentrations of unknown samples were determined using the slope and intercept calculated by a linear regression analysis of the calibration curves. Method Validation. The analytical LOD for each analyte was calculated as 3s0. S0 was estimated as the y-intercept of the bestfit line of the plot of the standard deviation of the standards used in the calibration curve versus the concentration.21 The LOD and S0 for each analyte are presented in Table 2. To determine the recovery of the method, several blank urine samples from the homogenized urine pool were enriched with known amounts of the pesticide standards and processed using the sample preparation method described above. Additional urine samples from the same urine pool were processed concurrently, but these were enriched following the extraction, before the concentration step. The sample sets were compared to each other and the recovery of the pesticide metabolites was calculated as a percentage. This experiment was performed at two different concentrations, and the results are presented in Table 3. The ISTD added during the processing corrected for variability in instrumental response. The accuracy of the method was evaluated by enriching several urine samples from the homogenized blank urine pool with known amounts of standards, processing and analyzing the samples, and plotting the expected versus measured values. A linear regression analysis of the data was then performed. A slope of 1.0 is indicative of 100% accuracy. Because we used mass spectrometric detection, we were able to use isotope dilution, which corrects for the recovery of each sample. Chemically, the native and labeled analytes behave virtually identically. Each species can be quantified individually because each can be distinguished by its respective mass. Thus, (19) Ashley, D. L.; Bonin, M. A.; Cardinali, F. L.; McCraw, J. M.; Holler, J. S.; Needham, L. L.; Patterson, D. G. Anal. Chem. 1992, 64, 1021-1029. (20) Shealy, D. B.; Bonin, M. A.; Wooten, J. V.; Ashley, D. L.; Needham, L. L.; Bond, A. E. Environ. Int. 1996, 22, 661-675. (21) Taylor, J. K. Quality Assurance of Chemical Measurements; Lewis: Chelsea, MI, 1987; pp 79-82.
Figure 1. Calibration curve for atrazine mercapturate spiked into urine. The circles represent the data points and the line represents a linear regression analysis of the data.
the native and labeled analytes would be expected to have equivalent recoveries, and the ratio of native to labeled species could be used to compensate for the variable method recoveries. Human Exposure Studies. The method described here was used to assess human exposure to atrazine, 2,4-D, and malathion in nonoccupationally exposed populations. Urine samples were collected from 151 adult and children volunteers with no known exposure to the pesticides of interest. All protocols were reviewed and approved by a human subject review committee and complied with all institutional guidelines for the protection of human subjects. RESULTS AND DISCUSSION A calibration curve for atrazine mercapturate spiked into urine is presented in Figure 1. The circles represent the data points and the line represents a linear regression analysis of the data. The curve is linear from 0.5 to 50 µg/L. The R2 value for the linear
regression analysis is 0.991. Calibration curves for malathion diacid and 2,4-D were similar, with R2 values of 0.979 and 0.986, respectively. Figure 2 is a plot of the quality control samples for atrazine mercapturate spiked into urine. The target value was 8.0 µg/L. The mean value established in multiple runs was 9.00 µg/L. The upper and lower 95th and 99th control limits are indicated on the plot. Two criteria were used to determine whether an analytical run was “out of control”: (1) Any QC sample outside of the 99th control limit indicated the analytical run was out of control, and (2) QC samples from the second of two consecutive analytical runs whose values were outside of the 95th control limits indicated that the second analytical run was out of control. As indicated in Figure 2, during several months of using the method, no analytical runs were deemed out of control. Figure 3 is a plot of the expected versus actual atrazine mercapturate concentrations (in µg/L). The circles represent the data points, and the line represents a linear regression of the data. The slope of the linear regression line is 1.0, indicating excellent agreement between the expected and measured concentrations for atrazine mercapturate in the samples. This same experiment was repeated for malathion diacid and 2,4-D. The slopes of linear regression fits to plots of the expected versus actual values for these analytes are 0.99 and 1.0, respectively, indicating that the accuracy of the measurement is high. The use of HPLC combined with MS/MS imparts a high degree of specificity to the analysis. The analytes and possible interferences are chromatographically separated prior to mass spectrometric analysis. Following ionization in the mass spectrometer, only the mass of interest (precursor ion mass) is allowed to pass into the collision cell, and in the last quadrupole, only the corresponding fragment ion (product ion mass) is allowed to pass to the detector (Table 1). A contaminant would have to (1) coelute on the HPLC, (2) have a precursor mass within 0.7 amu of the analyte mass, and (3) have a product ion mass within 0.7 amu of the product ion mass to interfere with the analyses. For the analysis of 2,4-D, a confirmation ion was also monitored. It was
Figure 2. Plot of the atrazine mercapturate QC samples at the lower spiked concentration. This plot is representative of the QC plots of all analytes.
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Figure 3. Plot of the expected versus actual atrazine mercapturate concentrations (in µg/L (ppb)), in urine. The circles represent the data points and the line represents a linear regression analysis of the data. The slope of the linear regression line is 1.0.
necessary for both the quantification and confirmation ion to be present in the proper ratios for the analyte to be considered present. In addition, the 2,4-D precursor and product ions both contain chlorine atoms, which produce a distinctive spectral fingerprint resulting from the naturally occurring 37Cl isotope. Monitoring the ions that contain the chlorine atoms further increases the specificity of the analysis for 2,4-D. In our laboratory, atrazine mercapturate has only been observed in two samples from the general population, although atrazine is the most widely used pesticide (by volume) in U.S. agricultural crop production;4 however, we have observed urinary atrazine mercapturate concentrations indicative of chronic/acute exposures in urine of people occupationally exposed to atrazine, indicating that our method is viable.9 These observations may indicate that incidental exposures to atrazine result in urinary atrazine mercapturate concentrations that are lower than 0.53 µg/ L. To accurately measure atrazine mercapturate in urine from the general population apparently requires lower method LODs. For the method described here, the analytical LODs for atrazine mercapturate may be improved upon if the recovery were improved from its current 26%. It is important to note that the extraction conditions used here are general conditions, which are necessary because of the structural diversity of the three analytes. The general extraction conditions most likely contributed to the low recovery for atrazine mercapturate. In a previous study, our laboratory analyzed samples from the general population to obtain a reference range of various pesticide
3530 Analytical Chemistry, Vol. 71, No. 16, August 15, 1999
metabolite urinary concentrations.3 The concentration of 2,4-D at the 95th percentile was 1.8 µg/L, indicating that most of the general population had urinary 2,4-D concentrations less than 1.8 µg/L; however, the method used had a limit of detection for 2,4-D of 1 µg/L, resulting in a detection frequency of 12%. The method reported here has a detection limit of 0.4 µg/L for 2,4-D. This is significantly below the concentration of the 95th percentile reported earlier, indicating that the method reported here is suitable for the measurement of urinary 2,4-D concentrations resulting from incidental exposure. We have applied the method described here to various studies and have observed detection frequencies of 54% as a result of the improved limit of detection. In a previous study of nonoccupationally exposed people, the frequency of detection for malathion diacid was 0.5%.15 The limit of quantification was 30 µg/L. Using the method reported here, we have observed a detection frequency of 51% for various studies, which is a result of our detection limit of 0.5 µg/L. For example, we have observed that 19% of the samples analyzed using our method had malathion diacid concentrations less than or equal to 1 µg/L. These concentrations would have been undetectable in the previously reported study. CONCLUSIONS We have developed an isotope dilution HPLC/MS/MS method for quantifying atrazine mercapturate, malathion diacid, and 2,4dichlorophenoxyacetic acid in urine. The limits of detection are approximately 0.5 µg/L (ppb) for each analyte in 10 mL of urine. Use of the method over several months has indicated that it is reliable and rugged, as evidenced by the control limits of the QC samples and the QC plot. The method described here is suitable for routine use in laboratories equipped with HPLC/MS/MS capabilities. The analytical LODs indicate that the method is suitable for quantifying the pesticide metabolites in occupationally exposed populations and for quantifying 2,4-D and malathion diacid in the general population. It is anticipated that the method will be suitable for quantifying atrazine mercapturate in the general population once the recovery is improved. The use of trade names is for identification purposes only and does not constitute endorsement by the Public Health Service, the Department of Health and Human Services, or the Centers for Disease Control and Prevention.
Received for review February 10, 1999. Accepted May 21, 1999. AC990130U
