JARDIM, I. C. S. F.

POZZEBON, J. M.

QUEIROZ, S. C. do N. de

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Determination of Herbicides by LC–MS/ESI 105105From: Methods in Biotechnology, Vol. 19, Pesticide ProtocolsEdited by: J. L. Martínez Vidal and A. Garrido Frenich © Humana Press Inc., Totowa, NJ8Determination of Herbicides in Human Urine by LiquidChromatography–Mass Spectrometry With ElectrosprayIonizationIsabel C. S. F. Jardim, Joseane M. Pozzebon, and Sonia C. N. QueirozSummaryA method for the determination of triazines (simazine, atrazine) and their metabolite2-chloro-4,6-diamino-1,3,5-triazine by liquid chromatography–mass spectrometry withelectrospray ionization (LC–MS/ESI) in human urine is described. The method outlinesthe sample preparation, which involves protein precipitation with acetonitrile and solid-phase extraction using C18 cartridges, and the qualitative and quantitative chromato-graphic analyses. The method may be used to assess occupational exposure to triazineherbicides following the urinary excretion of low levels of both the parent compoundsand their metabolites.Key Words: Atrazine; 2-chloro-4,6-diamino-1,3,5-triazine; human urine; liquid chro-matography–mass spectrometry with electrospray ionization; simazine; solid-phaseextraction; triazines.1. IntroductionOccupational exposure to triazine herbicides results in urinary excretion of lowlevels of both the parent compounds and their metabolites (1–6). Quantitative andqualitative analyses require highly sensitive and specific purification and determina-tion procedures.The increasing use of liquid chromatographic (LC) methods for pesticide analysisis because of their applicability in determining thermally labile compounds and polarcompounds, which require derivatization before gas chromatographic (GC) analysis(7). The good compatibility of aqueous samples with reversed-phase liquid chroma-tography contributes to the fact that LC is frequently applied for analyses of environ-mental and biological fluid samples. Although ultraviolet (UV) detectors are the mostcommon choice for LC, on-line liquid chromatography with mass spectrometry (LC–MS) is an interesting approach because conventional LC detectors can cause false-106 Jardim, Pozzebon, and Queirozpositive results. This technique combines the advantages of coupling the separationpower of LC to the unequivocal identification potential of MS for the determination ofpesticides in biological samples (8,9).This work describes a method for the determination by liquid chromatography–mass spectrometry with electrospray ionization (LC–MS/ESI) of the triazines simazineand atrazine and their metabolite 2-chloro-4,6-diamino-1,3,5-triazine (Fig. 1), presentin human urine.2. Materials1. Standard grade simazine (>98%), atrazine (>97%), and the metabolite 2-chloro-4,6-diamino-1,3,5-triazine (>96%).2. Methanol, pesticide grade.3. Acetonitrile, HPLC grade.4. Chloroform, pesticide grade.5. Water purified by a Milli-Q Plus System (Millipore, Bedford, MA).6. Envi C18 Supelclean 3-mL extraction tubes (e.g., Supelco, Bellefonte, PA).7. Urine samples. These must be kept in the freezer at –20°C until use. The samples arestable for 15 d (see Note 1).8. LC–MS system equipped with an injector with a 10-µL loop and a UV detector (e.g.,Waters, Milford, MA).9. Chromatographic column (150 × 3.9 mm id) and guard column (20 × 3.9 mm id) C18(e.g., Waters Nova-Pak C18, 4 µm).10. Vacuum extraction system (manifold) (e.g., Supelco).3. MethodsThe steps described below outline (1) sample preparation and (2) chromatographic analysis.3.1. Sample PreparationUrine samples are frozen at –20°C until use. After the urine samples are thawed,they must be shaken for homogenization. The required volume must be sampled asquickly as possible to avoid sedimentation of solids.Fig. 1. Chemical structures of the herbicides studied.Substituents in positions 4 and 6Herbicide R1 R22-chloro-4,6-diamino-1,3,5-triazine -H -H(metabolite)Simazine -C2H5 -C2H5Atrazine -C2H5 -CH(CH3)2Determination of Herbicides by LC–MS/ESI 1073.1.1. Removal of Proteins1. Take a 2-mL volume of urine.2. Basify the sample by addition of 200 µL of NH4OH (0.01%) (pH ∼9.0) and add 4 mL ofacetonitrile at room temperature.3. Centrifuge the mixture (5 min, 3000g).4. Take a 3-mL aliquot of the supernatant containing urine–acetonitrile (1:2 v/v) and dilutewith 20 mL Milli-Q water (see Note 2).3.1.2. Cleanup Using Solid-Phase ExtractionThe cleanup is made by solid-phase extraction using Envi C18 Supelclean extrac-tion tubes (3 mL) and a vacuum extraction system (manifold). The steps that followare involved.3.1.2.1. SORBENT CONDITIONINGRinse the cartridge with 10 mL methanol and then with 5 mL Milli-Q water for equili-bration. Avoid allowing the sorbent to dry; otherwise, the recovery will be decreased.3.1.2.2. SAMPLE APPLICATIONApply all the diluted sample (Subheading 3.1.1.) to the cartridge, under vacuum, ata flow rate of 3 mL/min.3.1.2.3. WASHING/REMOVAL OF INTERFERENCESRemove undesired matrix components by passing 5 mL Milli-Q water through the car-tridge. This eluate is discarded, and the sorbent bed is then dried under vacuum for 1 min.3.1.2.4. ELUTION AND CONCENTRATION OF THE SAMPLEWipe the delivery needles of the manifold and place labeled collection tubes underthe cartridges. With the vacuum off, add the elution solvent, 3 mL chloroform, to eachcartridge (see Note 3). Turn on vacuum and carefully open the tap of the manifold toinitiate elution of the analyte. The elution must be carried out slowly to obtain suitablerecovery of the analyte.The organic layer is evaporated to dryness under a stream of nitrogen, and the resi-due is dissolved in 200 µL of acetonitrile. The preconcentration factor is fivefold be-cause the volume applied to the cartridge corresponds to 1 mL of initial urine sample,and the final volume is 200 µL.3.2. Chromatographic Analysis3.2.1. Standard Solution PreparationStock solutions of simazine, atrazine, and the metabolite 2-chloro-4,6-diamino-1,3,5-triazine are prepared in methanol at concentrations of 100 µg/mL. Intermediateconcentrations are prepared in mobile phase at concentrations of 1000 µg/L of eachherbicide (see Note 4). These solutions are stored in the refrigerator at 4°C, at whichthey are stable for at least 60 d. Further solutions at five different concentrations (5,30, 60, 100, and 200 µg/L) of each analyte are prepared daily for construction of theanalytical curve.108 Jardim, Pozzebon, and Queiroz3.2.2. Spiked SamplesBecause certified reference material is not available and there are insufficientsamples for intercomparison assays, method accuracy is determined using spikedsamples. Three different concentrations (40, 60, and 80 µg/mL) of each pesticide arespiked into known volumes of pesticide-free urine (blank) to calculate recovery. Afterspiking the samples, carry out procedures as in Subheadings 3.1.1. and 3.2.1.3.2.3. Chromatographic ConditionsThe mobile phase is acetonitrile:H2O (40:60 v/v) with the pH of the mobile phaseadjusted to 3.0 with 0.1% CH3COOH (see Notes 5 and 6). The mobile phase flow rateis set at 0.3 mL/min. The column is directly coupled to the inlet of a chromatographwith a quadrupole MS system operated using the ESI source. Measurements are car-ried out using the positive ESI mode. All measurements are carried out at room tem-perature (see Note 7).For optimization of the MS parameters, each compound is dissolved in pH 3.0 mo-bile phase and injected separately. A source temperature of 150°C is used. Nitrogen isused as both nebulizer and drying gas at flow rates of 30 and 300 L/h, respectively.The capillary voltage is 25 V for the determination of these herbicides and theirmetabolite. For identification, the instrument is operated in the total ion mode; forquantification, acquisition is in the selected ion monitoring (SIM) mode. The selectedions for quantification are 145, 202, and 216 for 2-chloro-4,6-diamino-1,3,5-triazine,simazine, and atrazine, respectively (see Note 8).3.2.4. Qualitative and Quantitative Analyses (see Note 9)1. Inject the following sequence: solvent (mobile phase), matrix blank (urine without pesti-cide), standards, solvent, spiked samples (urine with spiked pesticides), and samples.Matrix blank, spiked samples, and samples are injected after processing according to thesteps in Subheadings 3.1.1. and 3.1.2. The standards, spiked samples, and samples are allinjected in triplicate.2. Compare the chromatograms. No peak should be detected at the retention times of thepesticides in the chromatograms of solvent and matrix blank.3. Determine the limit of detection (LOD) and the limit of quantification (LOQ).4. Construct the analytical curve with the areas obtained from the chromatograms vs con-centration. Linear analytical curves (r2  0.99) must be obtained in the range from LOQto 200 µg/L.5. Calculate the recoveries by comparing the replicate responses of extracted spiked sampleswith those of standards that represent 100% recovery. Recovery is calculated using thefollowing equation:Recovery (%) = (Mass of analyte after extraction × 100)/Mass of analyte added6. Calculate the precision in terms of repeatability using the results obtained from the recov-ery samples.7. If peaks are detected in the chromatograms of the samples, confirm the presence of theherbicide by comparing both retention time and the mass spectra using full-scan mode.8. Quantify the corresponding amount of the herbicide by the external standard method us-ing the linear equation obtained from the analytical curve.Determination of Herbicides by LC–MS/ESI 1094. Notes1. The stabilities of the herbicides in urine with storage time are evaluated by monitoringaliquots of urine having two different concentrations of simazine and atrazine (80, 150µg/L) and submitting them to replicate freeze/thaw cycles (10). The concentrations of theherbicides are determined after the initial thawing (zero time) and after thawing at suc-cessive prefixed times (once each week). Each concentration is compared with the meanof the zero time (initial concentration). Degradation values of <10% are obtained over aperiod of 15 d.2. The sample is diluted to decrease solution strength before the extraction procedure.3. The C18 Supelclean extraction tubes are eluted with methanol and compared with thoseeluted with chloroform. Recovery is generally good for both solvent systems, but back-grounds are cleaner using chloroform. The evaporation time for chloroform eluate is alsoshorter than that for methanol. Therefore, the use of chloroform as an elution solvent isrecommended for the triazines under study.4. An analytical curve prepared using mobile phase as solvent is preferable for dissolvingthe components of the sample, which is a prerequisite for using LC.5. The pH of the mobile phase is adjusted using a calibrated pH meter with glass and ther-mal compensation electrodes.6. A prerequisite in LC–MS is that the analyte must be ionizable in solution, so the mobilephase often contains a small amount of a volatile acid or base. If such additives impair thechromatographic separation, they can be added after the separation, before the eluententers the ESI interface. The separation of the triazine herbicides and their metabolite istested at various compositions of the eluent and at different pH values. Because of theirpolar character, the triazines do not interact strongly with the C18 reversed phase, themost utilized stationary phase in LC. Thus, it is necessary to add acetic acid to the mobilephase to increase the molecular ionization of the compounds, which also improves thedetection in analysis by MS.7. Quantification can also be performed by LC with UV detection. The UV detector is set at220 nm as a compromise between the maximum absorbance of the analytes and the re-duced background of the eluents at this wavelength (11). Acetonitrile is chosen as anorganic modifier in the mobile phase owing to its low absorbance background in the UVregion.8. After defining the conditions for SIM of the triazines, the product ions are recorded witha single quadrupole set at a fixed m/z value representing [M + H]+. A urine sample is acomplex matrix consisting of various components; therefore, it is essential to use anextraction procedure to remove these interferents. However, using SIM, no problem isencountered in the quantification of the analytes because only the ion corresponding toeach compound is selectively monitored.9. The method must be validated using the instrumentation with which the analyses will bemade. According to our results for validation of this method (12), analytical curves areobtained using standard solutions of the triazines, and they showed good linearity in therange from LOQ to 800 µg/L with correlation coefficients > 0.999. The following valuesare easily obtained for the validation parameters: average recoveries ranging from 82 to114%; of 0.4 µg/L LOD; 1.3 µg/L LOQ; and precision values of 0.5% (repeatability) and2.4% (intermediate precision). These values are considered acceptable for biologicalsamples (11).110 Jardim, Pozzebon, and QueirozAcknowledgmentsWe acknowledge FAPESP for financial support and a fellowship (to J. M. P.). Wealso thank C. H. Collins and W. Vilegas for helpful discussions and suggestions.References1. Mendas, G., Drevenkar, V., and Zupancic-Kralj, L. (2001) Solid-phase extraction withstyrene-divinylbenzene sorbent for high-performance liquid or gas chromatographic de-termination of urinary chloro- and methylthiotriazines. J. Chromatogr. A 918, 351–359.2. Catenacci, G., Barbieri, F., Bersani, M., Fereoli, A., Cottica, D., and Maroni, M. (1993)Biological monitoring of human exposure to atrazine. Toxicol. Lett. 69, 217–222.3. Mendas, G., Tkalcevic, B., and Drevenkar, V. (2000) Determination of chloro- andmethylthiotriazine compounds in human urine: extraction for gas chromatographic analy-sis with nitrogen-selective and electron capture detection. Anal. Chim. Acta 424, 7–18.4. Perry, M. J., Christiani, D. C., Mathew, J., Degenhart, D., Tortorelli, J., Strauss, J., andSonzogni, W. C. (2000) Urinalysis of atrazine exposure in farm pesticide applicators.Toxicol. Ind. Health 16, 285–290.5. Catenacci, G., Maroni, M., Cottica, D., and Pozzoli, L. Assessment of human exposure toatrazine through the determination of free atrazine in urine. Bull. Environ. Contam.Toxicol. 44, 1–7.6. Barr, D. A. and Needham, L. (2002) Analytical methods for biological monitoring of ex-posure to pesticides: a review. J. Chromatogr. B 778, 5–29.7. Hogendoorn, E. and van Zoonen, P. (2000) Recent and future developments of liquid chro-matography in pesticide trace analysis. J. Chromatogr. A 892, 435–453.8. Careri, M., Mangia, A., and Musci, M. (1996) Applications of liquid chromatography-mass spectrometry interfacing systems in food analysis: pesticide, drug and toxic sub-stance residues. J. Chromatogr. A 727, 153–184.9. Henion, J., Brewer, E., and Rule, G. (1998) Sample preparation for LC/MS/MS: analyzingbiological and environmental samples. Anal. Chem. 70, 650A–656A.10. Causon, R. (1997) Validation of chromatographic methods in biomedical analysis. View-point and discussion. J. Chromatogr. B 689, 175–180.11. Sacchero, G., Apone, S., Sarzanini, C., and Mentasti, E. (1994) Chromatographicbehaviour of triazine compounds. J. Chromatogr. A 668, 365–370.12. Pozzebon, J. M., Vilegas, W., and Jardim, I. C. S. F. (2003) Determination of herbicidesand a metabolite in human urine by liquid chromatography–electrospray ionization massspectrometry. J. Chromatogr. A 987, 375–380.

Determination of herbicides in human urine by liquid chromatography-mass spectrometry with electrospray ionization.

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Determination of herbicides in human urine by liquid chromatography-mass spectrometry with electrospray ionization.

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