1. John D. Vargo 1 ; Michael Schueller 1 ; Mary Skopec 2 1 State Hygienic Laboratory at the University of Iowa, Coralville, IA; 2 Iowa Department of Natural Resources, Des Moines, IA Determination of Emerging Contaminants in Iowa Surface Water at Low PPT Levels Using Direct Injection and SPE LCMSMS Overview The goals of this study were to:  Develop a direct aqueous injection (DAI) LCMSMS method to provide rapid and cost-efficient screening of targeted emerging contaminants at low PPT (ng/L) levels in surface water.  Compare the developed DAI LCMSMS method with an in-house procedure that uses solid phase extraction (SPE).  Evaluate the use of labeled internal standards to correct for instrument drift and/or matrix effects.  Test for emerging contaminants in Iowa surface water using both analytical procedures and compare results. Findings:  A DAI LCMSMS procedure was developed that permitted detection and quantification of emerging contaminants at low PPT levels.  The use of labeled internal standards effectively corrected for instrument drift and/or matrix effects.  Most analytes could be detected and quantified at a level of 0.01 µg/L by the DAI procedure and at 0.001 µg/L using the SPE extraction procedure. Introduction There is increasing interest for testing water for non-regulated contaminants, such as antibiotics, pharmaceuticals, endocrine disrupting chemicals, and others. Existing analytical methods, such as EPA 1694, tend to be labor-intensive, expensive to perform, and optimized for select classes of contaminants. There is a need for extraction procedures that are more generic in nature and accountable for many of the contaminants, as well as a need for a direct aqueous injection procedure that is rapid and cost-efficient to perform. LCMSMS testing methods are often prone to issues with instrument signal drift and/or matrix effects, especially when electrospray interfaces are used. There is a need for suitable internal standards that can appropriately correct for such issues. In this study, we contrast a generic SPE extraction procedure that was developed in our lab with a newly-developed direct aqueous injection (DAI) procedure. The effectiveness of labeled internal standards was also evaluated. Spike studies were conducted with surface water samples to examine: (1) effectiveness of the SPE extraction procedure, and (2) effectiveness of selected internal standards to correct for instrument signal drift and/or matrix effects. Finally, emerging contaminants were monitored upstream and downstream of Cedar Rapids and Iowa City using both the DAI and SPE procedures. Methods Sample Collection Surface water samples were collected in January, 2014, upstream (US) and downstream (DS) of Cedar Rapids (Cedar River) and Iowa City (Iowa River). Samples were collected in pre-cleaned amber glass bottles and stored at 2- 6⁰ C. No preservatives were used. Samples were extracted/analyzed within 7 days of collection. SPE Extraction 100 mL of water sample (400 mg of sodium bicarbonate added) was passed through a Waters Oasis HLB cartridge (200 mg) and subsequently eluted using 10 mL of methanol. Methanol was removed using N2 and a water bath at 40⁰ C until approximately 0.5 mL of aqueous remained. 0.5 mL of methanol was then added to each sample followed by vortex mixing and transfer to a 10-mL volumetric flask. 0.05 mL of internal standard mix was added followed by dilution to 10 mL using purified, organic-free water. QC Spikes for SPE Extracts The efficiency of the SPE extraction procedure was monitored by evaluating analyte recoveries from lab water samples (0.01 and 0.1 µg/L spiking levels) and field samples (0.1 µg/L) spiked with a mixture of the test analytes just prior to extraction. Standard addition spikes (0.1 µg/L ) were made to final extracts for evaluation of matrix enhancement/suppression effects in extracted samples. Direct Aqueous Injection (DAI) Sample was transferred to a 10-mL volumetric flask until the amount was near the calibration mark. 0.05 mL of internal standard was added. Additional sample was added to reach the 10 mL calibration mark followed by thorough mixing of the contents. Standard addition spikes (0.1 µg/L) were made to duplicate samples to evaluate matrix effects as well as assess how well internal standards were correcting residue amounts for each analyte. Analysis Sciex API 4000 QT instrument with electrospray interface Column – Zorbax SBC8, 3.5 µm, 3 x 150 mm Column heater temperature: 55⁰ C Acetonitrile/methanol/water (0.15% in acetic acid) gradient @ 0.5 mL/min 100 µL injection volume Positive and negative ions were monitored using data collection periods (system used in study did not have rapid polarity switching to permit simultaneous monitoring of positive and negative ions.) Positive ions: acetaminophen, caffeine, carbamazepine, cotinine, lincomycin, metoprolol, sulfadimethoxine, sulfamethazine, sulfamethoxazole, sulfathiazole, trimethoprim Negative ions: bezafibrate, diclofenac, gemfibrozil, ibuprofen, naproxen, triclosan Internal standards: acetaminophen-D4, caffeine-13C3, carbamazepine-D10, diclofenac-D4, metoprolol-D7, sulfamethoxazole-13C6 (1 µg/L in final sample and calibration standards) Results and Discussion Quantitation Quantitation was achieved via external calibration using standards ranging from 0.005 – 2.5 µg/L for most analytes. A quadratic curve fit with 1/x weighting was used. Analyte peak area responses were normalized to the response of the corresponding labeled internal standard. Excellent curve fit was observed for all analytes. A qualitative MRM ion pair was monitored for most analytes to confirm positive detections as well as verify the amount detected (± 30%). Amount Found (µg/L) Direct Aqueous Injection/SPE Spike Recoveries Iowa River Downstream ISTD Used Cedar River Upstream Cedar River Downstream Iowa River Upstream Iowa River DownstreamDAISPE Std Add. Acetaminophen1<0.010/0.0051<0.010/0.0034<0.010/0.00240.014/0.01510096102 Bezafibrate4<0.010/<0.0010 998692 Caffeine2<0.080/0.064<0.080/0.058<0.050/0.00880.10/0.1096100112 Carbamazepine30.012/0.0130.013/0.014<0.010/0.00630.027/0.02610199101 Cotinine1<0.010/0.0085<0.010/0.0094<0.010/0.0058<0.010/0.009389110112 Diclofenac4<0.010/<0.0010<0.010/0.010<0.010/<0.0010<0.010/0.010110102 Gemfibrozil4<0.010/0.0120.033/0.034<0.010/0.00210.058/0.061105 79 Ibuprofen4<0.016/0.014<0.010/0.010<0.010/<0.00100.017/0.01810490104 Lincomycin2<0.010/0.0085<0.010/0.00760.018/0.0170.018/0.016120129126 Metoprolol50.015/0.0120.022/0.019<0.010/<0.00100.035/0.0361039396 Naproxen40.023/0.0260.023/0.024<0.010/0.00180.025/0.0221008498 Sulfadimethoxine6<0.010/<0.0010 <0.010/0.0022 108106107 Sulfamethazine6<0.010/0.0034<0.010/0.0025<0.010/<0.0010 104100105 Sulfamethoxazole60.073/0.0710.073/0.0730.026/0.0260.12/0.129594- Sulfathiazole6<0.010/<0.0010 106102106 Triclosan4<0.25/<0.025 <0.10/<0.025 1297174 Trimethoprim20.013/0.0130.016/0.013<0.010/0.00240.050/0.038125126109 Matrix effects were minimal for samples analyzed by DAI but were more significant with SPE-extracted samples, no doubt due to concentration of dissolved organic matter during the extraction process. The labeled internal standards effectively corrected calculated residue concentrations in surface water samples. Spike recovery data for the SPE extraction procedure for surface water (Table 1) and organic-free lab water (data not presented here) showed very good, quantitative recovery for all of the test analytes with the exception of triclosan which had a recoveries of about 70%. DAI does not have issues with analyte extraction recovery. Low-level amounts of caffeine and triclosan were observed in all injected samples (including solvent blanks.) The reporting limits for these analytes were increased 5X times the observed background contamination level. There was very good agreement between the two procedures for detected contaminant concentrations in surface water samples. Positive detects for some analytes were observed and reported using the SPE extraction procedure that were below the detection limit for DAI. While the SPE extraction procedure is more sensitive, it comes at the trade-off of increased time/expense for labor and consumable supplies. The direct aqueous injection method that we developed was rapid, did not require sample prep time/expenses beyond addition of the labeled ISTDs to each sample, and was capable of quantifying most analytes (including confirmation ion pairs) at levels as low as 0.01 µg/L. The newest generation of more-sensitive LCMSMS systems should permit detection limits to be lowered an additional factor of 5X for most positive ions and perhaps up to 2-3X for negative ions. (Note: our evaluation of the newest generation of LCMSMS systems shows that while positive ion sensitivity is greater, many systems have difficulty matching the negative ion sensitivity that we can reach with our current system.) Peak Area ISTD Sample/Avg Peak Area ISTD Cal Std Iowa River DS DAI Iowa River DS SPE ISTD Number Acetaminophen-D4114751 Caffeine-13C394802 Carbamazepine-D1098913 Diclofenac-D492904 Metoprolol-D7102855 Sulfamethoxazole-13C692786 Table 1. Table 2.