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Chester R. Lapeza, Jr., * Donald G. Patterson, Jr., and John A. Liddle
Toxicology Branch, Clinical Chemistry Division, Center for Environmental Health, Centers for Disease Control, U.S. Department of Health and Human Services, Atlanta, Georgia 30333
An apparatus is described for the automation of the first part of a proven method for part-per-trillion dioxin analysis. A microprocessor controls the valves and solvent flow for the sequential extraction and enrichment of five tissue samples over a 20-h period. Sample and solvent selection for the multicolumn system and reversed flow elution, fraction collection, and column regeneration are all accomplished without operator attention. The time required by the analyst per sample has been reduced 50% compared with the totally manual method, resulting in greater throughput and lower personnel costs. The apparatus can be easily assembled with commercially available components.
In recent years interest in the analysis of tissue samples for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has rapidly increased , but an accurate determination at the part-per-trillion level presents many problems to the public health toxicologist. The toxicity of the analyte and the required internal standard, the extraction of polychlorinated dibenzo-p-dioxins (PCDDs) and related compounds from the biological matrix, the removal of interfering polychlorinated biphenyls (PCBs) and pesticide residues (often present at parts-per-million levels) 
Figure 1. Apparatus used to automate extraction and cleanup part of method for the analysis of tissue samples for 2,3,7,8-tetrachlorodibenzo-p-dioxin. of human adipose was based on the method developed by Smith et al. as a modular approach to sample cleanup that could be easily automated . This method is carried out in three stages: extraction and cleanup, fractionation of residues, and analysis by high-resolution gas chromatography/mass spectrometry (GC/MS). The apparatus used to automate the first part of the method is described here.
EXPERIMENTAL SECTION
Apparatus. The entire system is constructed of commercially available components and requires minimal mechanical skills to set up and operate (see Figure 1). The samples and their extracts contact only glass and Teflon, thereby minimizing the possibility of contamination. The carbon/glass fiber and potassium silicate/silica gel columns are reusable and are regenerated through solvent washings during the automated run.
All valves, containers, and columns are interconnected with flanged tubing made of Teflon and threaded polypropylene bushings and couplings (Universal Scientific, Atlanta, GA). Solvents (Burdick and Jackson) are pumped directly from the 4-L bottles in which they are shipped by the Fluid Metering, Inc., Model RP-SY-ICSC pump, fitted with a micrometer low-flow adjustment kit. High-pressure liquid chromatography, low-pressure solvent filters may be used, and they also serve as weight to maintain the pickup of solvent from the bottom of the container. Because solvent volumes are controlled by the flow set at the pump (5.0 mL/min), air bubbles must be kept out of the system and especially out of the pump head where they could become trapped, allowing little, if any, solvent to pass. This can be prevented by (1) locating the pump below the level of the solvent reservoirs. (2) purging the system of air before starting the pump, and (3)
including bubble traps. The bubble traps are the same columns as those used for the carbon column and are attached to the outlet of the sample column with an 11-mm coupling (5805–15).
The three glass ace-thred columns are described as follows: sample extraction column (600 × 50 mm 5795–54); silicate column (5796–34 precolumn); carbon column and bubble traps (11 mm custom 5795–823); and threaded, end fitting adapters made of Teflon (5801–14) are supplied by Ace Glass, Inc., Vineland, NJ.
Rheodyne low-pressure valves made of Teflon are used to control flow directions and to select samples and solvents for each step of the procedure. Three configurations of the valves are required: six-position rotary (three each), four-way rotary, and a tandem six-position rotary. All are pneumatically actuated and controlled by 120-V ac solenoid valves.
The multisample, multistep enrichment process (4) is automatically controlled through the use of an Eldex Laboratories (Menlo Park, CA) Chromat-A-Trol programmable microprocessor. This unit is easily programmed with up to 250 instructions (1.25K RAM with battery protection) and allows files to be looped or stacked, programs to be altered in progress, and any function to be performed manually at any time. Current channel status and elapsed run time are displayed at all times on the control panel. The unit is programmed to turn the pump on and off and to supply voltage to each valve’s controlling solenoid through the accessory ac power module. The Chromat-A-Trol also provides for selection of each sample, through the Eldex selector valve, without pneumatic interfacing.
Operation. The Chromat-A-Trol is preprogrammed to control the switching of valves and pumping of selected solvents for up to five samples (1/file). At time 0, power is applied to the pump, and the selector valve is then set to receive sample extract from the first sample column. As the program progresses (time being determined by the amount of solvent pumped at 5 mL/min), the silicate column is removed from the flow, the carbon column is washed and then eluted in the reverse direction, and finally, the carbon and silicate columns are regenerated with multiple solvent washings. The pump is then turned off, or if more samples are to be extracted, the selector valve is switched to the next position and the cycle is repeated—all under the control of the Chromat-A-Trol (a sample program can be obtained upon request from the authors).
Preparation for a run of five samples is as follows. The four solvent containers are filled with appropriate amounts of solvents, and any air in the lines is bled by pulling the solvent through the vent valve with a gas-tight syringe. Next, the silicate columns are packed (or judged reusable), and the 50/50 hexane/methylene chloride solvent mix is pumped through the system to remove any air. The sample columns are then packed with adsorbents, and the samples are prepared for extraction (see ref 4). The first sample may then be loaded into the extraction column. With the selector valve set for the appropriate column and the vent valve open, air is allowed to escape the system until the first of the extraction solvent approaches the sample valve. The valve is then advanced to the next column, and its sample is loaded and the lines are bled as before. Finally, 500 mL of the 50/50 solvent mix is added to each extraction column. Before the program is begun, the valves must be in their starting positions, the solvent reservoirs full, and the collection vessels in place. With all samples loaded and all air removed from the system, the program is ready to take over and complete the extraction of the five samples without further attention. In its present configuration, the system can handle five adipose samples from initial homogenization through enrichment of planar trace-level environmental contaminants over a period of 20 h.
At this point the enriched extract, in 50 mL of toluene, must be handled manually (4) to exchange the solvent for hexane and be carried through the final chromatography steps before being analyzed by high-resolution GC/MS. Further development may allow for the removal of solvent to be automated, or the procedure may be changed so that the final separation can be included without solvent exchange.
RESULTS AND DISCUSSION
Performance Evaluation. The system has been tested for false positives, carryover from sample to sample, and precision, and it has been found to perform as well as its manual counterpart. A system blank consisting of all adsorbents, solvents, and internal standard but no tissue has been included in each set of five samples processed. None of these blanks analyzed would have met our criteria (4) for being reported as positive for TCDD. Possible carryover was evaluated by placing 1200 pg of 13C-labeled 2,3,7,8-TCDD (our internal standard) in sample positions 1, 2, and 4 and processing positions 3 and 5 as blanks. Analysis of the two blanks showed no detectable 2,3,7,8-TCDD, indicating no anticipated carryover from a 10-g adipose sample containing up to 120 pptr (parts per trillion) 2,3,7,8-TCDD.
Precision of the method was determined by comparing results of dioxin analysis obtained following either manual or automated cleanup (Table I). Samples were either human adipose or spiked animal fat from our quality control (QC) pools.
The automated system has been accepted for routine use at CDC and has been used for processing samples from several cases of possible dioxin exposure. Our routine analytical run consists of five samples, including one sample from our QC pool (high, medium, or low), and four unknowns.
Our plans for future development of the system include modification or replacement of the control module so that the pneumatic valves can be replaced with electronic ones like the selector valves. In addition, we may be able to modify the procedure to eliminate the need for a solvent exchange, thereby allowing the alumina chromatography step to be included in the automation. Because the system is modular and very flexible, the apparatus could perhaps be adapted to other applications that require multicolumn chromatography cleanup or multisolvent elution, or both.
The advantages of automating this or any labor-intensive procedure are many. Tedious, repetitive manipulations are performed mechanically, reducing the analyst’s boredom and exposure to hazardous substances while increasing the procedure’s precision and reliability. Complete runs can be set up to process sequentially QC and analytical samples. Previously, only two samples per 8-h day were processed (on two separate apparatuses), but now five samples can be processed automatically overnight, resulting in more than a 50% reduction in the time required of the analyst per sample.
ACKNOWLEDGMENT
We thank Louis Alexander and Ralph O’Connor for their analytical support and especially David Griffin, of Universal Scientific, Inc., for his invaluable assistance in engineering and selecting compatible components.
Registry No. TCDD, 1746–01–06.
LITERATURE CITED
RECEIVED for review September 27, 1985. Accepted November 19, 1985. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.
Agency for Toxic Substances and Disease Registry, 1825 Century Blvd, Atlanta, GA 30345
Contact CDC: 800-232-4636 / TTY: 888-232-6348
