Anal. Chem. 2003, 75, 723-730
Combination of High-Performance Liquid Chromatography and Microplate Scintillation Counting for Crop and Animal Metabolism Studies: A Comparison with Classical On-Line and Thin-Layer Chromatography Radioactivity Detection Michael Kiffe,* Andrea Jehle, and Robert Ruembeli
Crop and Animal Metabolism, Syngenta Crop Protection AG, CH-4002 Basel, Switzerland
Samples from crop or animal metabolism studies of pesticides were used to evaluate the performance of the combination of analytical or narrow-bore HPLC and microplate scintillation counting (TopCount). Samples with extreme matrix content such as grain and tomato extracts from crop metabolism studies as well as extracts from hen excreta or goat urine from farm animal metabolism studies could be injected, analyzed, and quantified directly without any sample pretreatment. The minimum amount of radioactivity injected was ∼1 Bq (60 dpm). Counting times from 5 to 60 min were used for detection and quantification. These results were compared with those from classical on-line radioactivity detection and with radioactivity detection on thin-layer chromatography plates.The combination of analytical or narrow-bore HPLC and microplate scintillation counting (TopCount) offers high sensitivity and high resolution power at the same time. It could be clearly demonstrated that the combination of HPLC with microplate scintillation counting is superior to the classical on-line radioactivity detection and at least equivalent to the classical thin-layer radiochromatography regarding performance and sensitivity. Crop and animal metabolism studies are essential requirements for the registration of new agrochemicals: metabolism studies in mammals and plants are crucial in the safety assessment process. The aim of crop metabolism studies is to get information on the fate of the compounds (metabolic degradation) in plants, as well as on the residue levels of the active compound and relevant metabolites present in food or animal feed, leading finally to a residue definition. Farm animal metabolism studies have to be conducted to cover the exposure of farm animals to pesticidetreated feed.1 * Corresponding author. E-mail:
[email protected]. Fax: +41 61 6964317. (1) Roberts, T. Metabolism of Agrochemicals in Plants, 1st ed.; John Wiley & Sons: Ltd.: Chichester, 2000; Chapter 1 and 3. 10.1021/ac020363c CCC: $25.00 Published on Web 01/21/2003
© 2003 American Chemical Society
The fate of agrochemicals can be followed most efficiently using isotope tracer technology. Due to the wide occurrence of carbon and due to aspects of radiation safety and stability, 14C is the preferred isotope compared to 3H and is also recommended in certain regulatory guidelines (e.g., EPA Residue Chemistry Test Guidelines, OPPTS 860.1300 Nature of the Residue-Plants, Livestock, EPA 712-C-96-172). The most important method for separation and quantification of metabolites and for the analysis of metabolic pathways of agrochemicals in biological systems is thin-layer chromatography (TLC).2-4 When this method is applied with radiolabeled compounds it is called thin-layer radiochromatography.5 Thin-layer chromatography is extremely flexible regarding the combination of stationary and mobile phases and it is fast, low-cost, efficient, and, due to more sophisticated detection systems for radioactivity, extremely sensitive. Radioactive zones can be detected directly in the same manner as ordinary autoradiography (e.g., Fuji BioImaging Analyzer BAS1000; Fuji Photo-Film Corp. Ltd., Tokyo, Japan) or indirectly by scraping off radioactive spots from the TLC plate followed by liquid scintillation counting. Thin-layer chromatography, especially based on silica gel, is a robust technology since even samples with high matrix content (e.g., extracts of wheat grains, apples, sugar beets, and feces) can be analyzed directly without significant loss of resolution. However, the measuring times are long. The use of high-performance liquid chromatography (HPLC) with on-line radioactivity detection in crop or farm animal metabolism studies for these types of extracts is in most cases more restricted. Extracts have to be pretreated by means of solidphase extraction or liquid-liquid partition in order to reach sufficient amounts of radioactivity for detection or to remove interacting matrix compounds, influencing the eluting behavior or destroying the stationary phase. However, pretreatment of extracts might not necessarily reflect the original metabolic pattern (2) Sherma, J. J. Chromatogr., A 2000, 880, 129-147. (3) Sherma, J. J. AOAC Int. 2001, 84, 993-999. (4) Ahmed, F. E. Trends Anal. Chem. 2001, 20, 649-661. (5) Fried, B.; Sherma, J. Handbook of Thin-Layer Chromatography; Chromatographic Science Series, 1st ed.; Marcel Dekker: New York, 1990; Vol. 55, Chapter 12.
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or can cause changes in the chemical composition of the metabolite pattern due to loss of radioactivity or chemical reactions. Therefore, the use of HPLC in combination with online radioactivity detection in many cases is not satisfactory for the analysis of low concentrations of radioactive residues. However, that classical method is very fast with regard to radioactivity counting and method development. Recently Boernsen et al.6 introduced the application of the microplate scintillation counter (TopCount microplate scintillation and luminescence count ) TopCount; Packard Instrument Co., Meriden, CT) in combination with capillary liquid chromatography or capillary electrophoresis. In principle, this system consists of an HPLC system able to run on low flow rates or a capillary electrophoresis system combined with a fraction collector for 96well plates, collecting fractions eluting from an HPLC column or a capillary with a makeup flow. The radioactivity in the 96-well plates is determined off-line with the TopCount. The TopCount system is able to perform fast and sensitive analysis using low amounts of radioactivity. This paper describes the experimental setup of the TopCount in combination with narrow-bore HPLC (2- or 2.1-mm i.d.) and analytical HPLC (4.6-mm i.d.) employing samples resulting from crop (extract of tomatoes and wheat grain as well as tomato cell extracts and paddy water of a paddy rice study) or farm animal studies (goat urine samples or extracts of hen excreta). The chosen samples are representative of crop or farm animal metabolism studies with medium to high matrix content. Comparison of the TopCount with classical HPLC using on-line radiodetection and thin-layer radiochromatography was done in order to get information regarding sensitivity and resolution for metabolite quantification. EXPERIMENTAL SECTION (a) Sample Preparation. The described crop and farm animal samples were obtained from metabolism studies after treatment with radiolabeled pesticides (A-D) (Chemical structures and names of A-D are confidential.) The specific activity of the applied radiolabeled compounds (14C) was in the range of 0.5-3.0 MBq/ mg (30 000-180 000 dpm/µg). Paddy water samples (14 days after application) from a rice metabolism study and urine samples from goat were injected directly into HPLC or applied directly onto TLC without any further purification. Tomatoes were homogenized in the presence of liquid nitrogen. The homogenate was extracted with acetonitrile/water, 80:20 v/v, using a mechanical shaker and centrifuged. The resulting extract was used for analysis. Spring wheat plants were separated into grain, husk, and straw. An aliquot of grain was then homogenized and extracted as described for tomatoes. The extract was used for further analysis by TLC, analytical HPLC, and narrow-bore HPLC. The cells from tomato cell cultures were separated from the medium by filtration and washed three times with distilled water. Harvested cells were homogenized in the presence of acetonitrile/ (6) Boernsen, K. O.; Foeckher, J. M.; Bruin, G. Anal. Chem. 2000, 72, 39563959.
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water, 80:20 v/v. The homogenate was centrifuged at 900013 500g to be re-extracted with acetonitrile/water. The cell media and extract were analyzed by TLC, analytical HPLC, and narrowbore HPLC without further purification. Hen excreta were homogenized and shaken with acetonitrile followed by extraction with acetonitrile/water, 80:20 v/v. The combined filtrates were reduced in volume to be injected directly. A 14C recovery test was performed for all extracts analyzed. The recovery test was done before and after each evaporation step. Evaporation and concentration of extracts were in general done on a rotary evaporator (temperature of water bath
