Depth-Profiling of Environmental Pharmaceuticals in Biological Tissue by Solid-Phase Microextraction Xu Zhang, † Ken D. Oakes, † Md Ehsanul Hoque, ‡ Di Luong, † Shirin Taheri-Nia, † Claudia Lee, † Brendan M. Smith, † Chris D. Metcalfe, ‡ Shane de Solla, § and Mark R. Servos* ,† † Department of Biology, University of Waterloo, Ontario, N2L 3G1, Canada ‡ Water Quality Centre, Trent University, Peterborough, Ontario, K9J 7B8, Canada § Wildlife and Landscape Science Directorate, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 * S Supporting Information ABSTRACT: The parallel in vivo measurement of chemicals at various locations in living tissues is an important approach furthering our understanding of biological uptake, transportation, and transformation dynamics. However, from a technical perspective, such measurements are difficult to perform with traditional in vivo sampling techniques, especially in freely moving organisms such as fish. These technical challenges can be well addressed by the proposed depth-profiling solid-phase microextraction (DP-SPME) technique, which utilizes a single soft, flexible fiber with high spatial resolution. The analytical accuracy and depth-profiling capability of DP-SPME was established in vitro within a multilayer gel system and an onion artificially contaminated with pharmaceuticals. In vivo efficacy was demonstrated by monitoring pharmaceutical distribution and accumulation in fish muscle tissue. The DP-SPME method was validated against pre- equilibrium SPME (using multiple small fibers), equilibrium SPME, and liquid extraction methods; results indicated DP-SPME significantly improved precision and data quality due to decreased intersample variation. No significant adverse effects or increases in mortality were observed in comparisons of fish sampled by DP-SPME relative to comparable fish not sampled by this method. Consequently, the simplicity, effectiveness, and improved precision of the technique suggest the potential for widespread application of DP-SPME in the sampling of heterogeneous biotic and abiotic systems. M onitoring drug distributions in animal tissues is important for pharmaceutical discovery and develop- ment, as well as from a toxicological perspective where an understanding of the uptake, pharmacokinetics, and biotrans- formation of drugs (in animal or human experimental subjects) is often required. For toxicologists, the measurement of waterborne organic contaminants (including pharmaceuticals and personal care products, PPCPs) in fish muscle is critical to understanding the toxicokinetics and potential impact on humans via the food chain. 1-7 Using traditional ex vivo or in vitro sampling procedures, a large number of animals must be sacrificed (due to significant interanimal variation) to obtain sufficient statistical power. 8 Mounting pressure (animal ethics, fiscal constraints) to reduce the number of experimental animals used, while simultaneously improving data quality, dictate that an in vivo sampling technique with good precision, moderate invasiveness, and insignificant lethality is desirable. For in vivo fish sampling, solid-phase microextraction (SPME) is a promising approach due to the simplicity and analytical sensitivity of the method relative to traditional in vivo sampling techniques such as microdialysis (MD) and ultra- filtration (UF). Sophisticated surgery is often required for MD or UF sampling, and several days are routinely required for wound recovery prior to sample collection. Both MD and UF require syringe pumps and power supplies, rendering these approaches cumbersome for field applications, 9-13 while MD cannot be easily used for depth-profiling analysis due to the unwieldy configuration of the probe. The in vivo SPME technique has been successfully employed to monitor the toxico-kinetics of pharmaceuticals and pesticides in rainbow trout (Oncorhynchus mykiss) dorsal-epaxial muscle. 14-16 Re- cently, the bioconcentration of several widely detectable PPCPs partitioning to muscle tissue was simultaneously monitored relative to that partitioning to the adipose fin using small segmented fiber coatings which improved spatial resolu- tion. 14,16 While the segmented fiber design introduced the potential for sampling heterogeneous sample systems using SPME, this technique precluded the use of single fiber coating probes (such as those available commercially) for spatially resolving analytes within heterogeneous sample systems. During our fish experiments, an important concern arose Received: February 16, 2012 Accepted: June 25, 2012 Article pubs.acs.org/ac © XXXX American Chemical Society A dx.doi.org/10.1021/ac3004659 | Anal. Chem. XXXX, XXX, XXX-XXX