The ability to quantify levels of target analytes in biological samples accurately and precisely in biomonitoring involves the use of highly sensitive and selective instrumentation such as tandem mass spectrometers and a thorough understanding of highly variable matrix effects. Typically, matrix effects are caused by co-eluting matrix components that alter the ionization of target analytes as well as the chromatographic response of target analytes, leading to reduced or increased sensitivity of the analysis. Thus, before the desired accuracy and precision standards of laboratory data are achieved, these effects must be characterized and controlled. Here we present our review and observations of matrix effects encountered during the validation and implementation of tandem mass spectrometry-based analytical methods. We also provide systematic, comprehensive laboratory strategies needed to control challenges posed by matrix effects in order to ensure delivery of the most accurate data for biomonitoring studies assessing exposure to environmental toxicants.

Atrazine [6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine] is the most widely used herbicide in the United States. In recent years, there has been controversy about atrazine's potential endocrine/reproductive and neurological adverse effects in wildlife and humans. The controversy triggered several environmental and epidemiologic studies, and it generated needs for sensitive and selective analytical methods for the quantification of atrazine, atrazine metabolites, and degradation or hydrolysis products. We developed a two-dimensional high performance liquid chromatography (2D-HPLC) method with isotope dilution tandem mass spectrometry detection to measure atrazine in urine, along with 11 atrazine metabolites and hydrolysis products, including 6-chloro (Cl), 6-mercapto (Mer) and 6-hydroxy (OH) derivatives, and their desethyl, desisopropyl and diamino atrazine analogs (DEA, DIA and DAA, respectively). The 2D-HPLC system incorporated strong cation exchange and reversed phase separation modes. This versatile approach can be used for the quantitative determination of all 12 compounds in experimental animals for toxicological studies. The method requires only 10mcL of urine, and the limits of detection (LODs) range from 10 to 50mcg/L. The method can also be applied to assess atrazine exposure in occupational settings by measurement of 6-Cl and 6-Mer analogs, which requires only 100mcL of urine with LODs of 1-5mcg/L. Finally, in combination with automated off-line solid phase extraction before 2D-HPLC, the method can also be applied in non-occupational environmental exposure studies for the determination of -6-Cl and 6-Mer metabolites, using 500mcL of urine and LODs of 0.1-0.5mcg/L.

Concern has increased about the resulting health effects of exposure to melamine and its metabolic contaminant, cyanuric acid, after infants in China were fed baby formula milk products contaminated with these compounds. We have developed a selective and sensitive analytical method to quantify the amount of cyanuric acid in human urine. The sample preparation involved extracting free-form cyanuric acid in human urine using anion exchange solid phase extraction. Cyanuric acid was separated from its urinary matrix components on the polymeric strong anion exchange analytical column; the analysis was performed by high performance liquid chromatography-tandem mass spectrometry using negative mode electrospray ionization interface. Quantification was performed using isotope dilution calibration covering the concentration range of 1.00-200ng/mL. The limit of detection was 0.60ng/mL and the relative standard deviations were 2.8-10.5% across the calibration range. The relative recovery of cyanuric acid was 100-104%. Our method is suitable to detect urinary concentrations of cyanuric acid caused by either environmental exposures or emerging poisoning events.

We report an improved solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry method with isotope dilution quantification to measure seven atrazine metabolites in urine. The metabolites measured were hydroxyatrazine (HA), diaminochloroatrazine (DACT), desisopropylatrazine (DIA), desethylatrazine (DEA), desethylatrazine mercapturate (DEAM), atrazine mercapturate (ATZM), and atrazine (ATZ). Using offline mixed-mode reversed-phase/cation-exchange solid-phase extraction dramatically increased recovery and sensitivity by reducing the influence of matrix components during separation and analysis. DACT extraction recovery improved to greater than 80% while the other analytes had similar extraction efficiencies as previously observed. Limits of detection were lower than our previous method (0.05-0.19ng/mL) with relative standard deviations less than 10%. The total runtime was shorter (18min) than the previous on-line method, thus it is suitable for large-scale sample analyses. We increased the throughput of our method twofold by using the newer extraction technique.

Human exposure to methyl parathion can be assessed by measuring the concentration of its metabolite paranitrophenol (PNP) in urine. Our biologic monitoring study in Chiang Mai, Thailand, measured PNP and dialkylphosphate metabolites (i.e., dimethylphosphate [DMP] and dimethylthiophosphate [DMTP]) of methyl parathion in urine samples collected from 136 farmers (age 20 to 65 years) and 306 school children (age 10 to 15 years) in 2006. Participants came from two topographically different areas: one was colder and mountainous, whereas the other was alluvial with climate fluctuations depending on the monsoon season. Both children and farmers were recruited from each area. Despite methyl parathion's prohibited use in agriculture in 2004, we detected PNP in >90% of all samples analyzed. We applied a nonparametric correlation test (PNP vs. DMP and DMTP) to determine whether the PNP found in most of the samples tested resulted from exposures to methyl parathion. DMP (Spearman's rho = 0.601 [p = 0.001] for farmers and Spearman's rho = 0.263 [p <0.001] for children) and DMTP (Spearman's rho = 0.296 [p = 0.003] for farmers and Spearman's rho = 0.304 [p<0.001] for children) were positively correlated with PNP, suggesting a common source for the three analytes, presumably methyl parathion or related environmental degradates. Although we found a modest correlation between the metabolites, our findings suggest that despite the prohibition, at least a portion (approximately 25% to 60%) of the PNP detected among farmers and children in Thailand may be attributed to exposure from continued methyl parathion use.