Chapter 15
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Determination of Veterinary Drugs in Dry Milk Powder by Supercritical Fluid Extraction—Enzyme-Linked Immunosorbent Assay Viorica Lopez-Avila and Janet Benedicto Midwest Research Institute, California Operations, 625-B Clyde Avenue, Mountain View, CA 94043 The objective of this study was to establish whether supercritical fluid extraction (SFE) coupled with enzyme-linked immunosorbent assay (ELISA) is a viable technique for the determination of sulfamethazine in dry milk powder at concentrations as low as 2.5 ng/g. Extraction and analysis of organic compounds in various matrices by SFE-ELISA has been under evaluation in our laboratory since it results in greater sample throughput, allowing rapid screening of environmental samples. In this study, we demonstrated that sulfamethazine could be extracted quantitatively (average recovery at 5-ng/g spike was 91.7 percent) from spiked dry milk powder at 450 atm and 80°C using supercritical carbon dioxide modified with 10 percent methanol as the extraction fluid. The extracted material was collected in methanol, concentrated to dryness, and redissolved in phosphate buffer for analysis by a competitive ELISA. The precision of the ELISA technique, as established over a period of 17 days, was 15 percent or better for sulfamethazine concentrations ranging from 5 to 15 ng/mL of extract in phosphate buffer. Analytical scale SFE has gained popularity among the conventional sample preparation techniques such as Soxhlet and sonication extraction because (a) it requires much less solvent for extracting the compounds of interest from the solid matrix, (b) is fast, (c) is selective, and (d) allows easy removal and disposal of the extraction solvent. The most common fluid in SFE is carbon dioxide, but small amounts of modifiers (e.g., methanol, acetonitrile, methylene chloride) can be added to supercritical carbon dioxide to make it more polar. Recently, we have seen a continuous increase in the number of publications dealing with analytical SFE and the number of compounds that can be extracted by SFE. For these reasons, we have undertaken this study to evaluate SFE as a potential extraction technique for sulfamethazine from powdered milk. 0097-6156/96/0636-0144$15.00/0 © 1996 American Chemical Society
In Veterinary Drug Residues; Moats, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
Downloaded by PENNSYLVANIA STATE UNIV on September 10, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0636.ch015
15.
LOPEZ-AVTLA & BENEDICTO
Veterinary Drugs in Dry Milk Powder
145
A study by Malik et al. (1) concluded that sulfamethazine could be extracted from 10-g samples of dry milk powder with supercritical carbon dioxide at 50°C and 390 atm; however, to achieve quantitative extraction efficiency the extraction time had to be extended to 2 hrs (the concentration of sulfamethazine in dry milk powder was 163 ng/g). Extraction of sulfonamides from food animal products (e.g., liver, swine muscle), fortified chicken tissue, and various solid supports (e.g., diatomaceous earth, silica gel) using carbon dioxide and carbon dioxide modified with methanol have been reported (2-4). To enhance the yield of sulfonamides extracted from solid supports with supercritical carbon dioxide, Tena and coworkers (4) used 0.1 M trimethylphenylammonium hydroxide in methanol, as ion-pair reagent, and extraction with supercritical carbon dioxide at 40°C and 280 atm. Another technique that is also gaining momentum, especially in environmental analysis, is ELISA. The most common format in enzyme immunoassays is competitive ELISA. In this type of assay, the antibodies specific to the analyte to be detected are immobilized (coated) onto a solid phase, either a plastic tube, a well on a microtiter plate, or paramagnetic particles. The enzyme-hapten conjugate and the target analyte are added to the antibody-coated tube or plate for a short incubation (15 to 30 min) during which time the target analyte and the enzymehapten conjugate compete for antibody binding sites. After incubation, the unbound material is removed, and a substrate-chromogen solution is added. After another short incubation period during which the enzyme converts the substrate-chromogen to a colored product, the reaction is terminated and the absorbance is measured with a spectrophotometer. The ELISA is attractive because it (a) allows high sensitivity and high degree of selectivity in antibody binding, (b) is relatively cheap, and (c) has potential for field use. There are, however, disadvantages to ELISA that need to be presented. The development of the ELISA technique is lengthy (i.e., takes 8 to 12 months), (b) the ELISA specificity limits its use to analysis of only one or a few closely related compounds, (c) the detection, range is very narrow, and (d) occasionally ELISA shows too many false positives or fails to detect the analyte (5). This paper will present the extraction of sulfamethazine from dry milk powder with supercritical carbon dioxide modified with methanol and detection of sulfamethazine by ELISA. Materials and Methods Reagents. All immunological reagents used in this study—including reaction tubes coated with anti-sulfamethazine antibody, horseradish peroxidase—sulfamethazine conjugate (traces), phosphate buffer (20 mM, pH 6+0.2) 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) and hydrogen peroxide in citrate buffer (color developer), and dilute sodium dodecyl sulfate (stopping solution) were obtained from Idetek, Inc. (Sunnyvale, CA). Details on the composition of the various reagents are not available (proprietary information). Sulfamethazine was obtained form Sigma Chemical Co. (St. Louis, MO). The dry milk powder used in this study was Carnation Nonfat milk and was purchased from a local supermarket.
In Veterinary Drug Residues; Moats, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
146
VETERINARY DRUG RESIDUES
Table I. Optimization of SFE Conditions for Sulfamethazine Percent Recovery
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Parameter 300 atm/60°C/30 min dynamic/carbon dioxide 450 atm/80°C/30 min dynamic/carbon dioxide 450 atm/120°C/30 min dynamic/carbon dioxide
Recovery not determined due to high background
450 atm/80°C/30 min dynamic/carbon dioxide with 10 percent methanol (spiked at 5 ng/g)
91.7 ± 17 (no TEA) (n=4) 105 (with 20 μL TEA) 68. l (with 40 TEA) a
a
350 atm/80°C/30 min dynamic/carbon dioxide with 10 percent methanol (spiked at 5 ng/g) 250 atm/80°C/30 min dynamic/carbon dioxide with 10 percent methanol (spiked at 5 ng/g) a
21 (n=2) 13 (n=2)
Single determinations. Table II. ELISA Performance (Sulfamethazine) Measured concentration (ng/mL)
Standard cone. (ng/mL) 0 5 7.5 10 15
~ I
2
3
0 4.96 — 11.3 12.7
0 4.27 — 8.67 11.3
0 4.72 5.91 12.0 12.2
Ave. ± SD
4
5
6
7
8
0 4.87 6.96 8.64
0 4.95 7.18 10.6 —
0 5.14 8.72 9.38 —
0 5.22 7.54 10.2 —
0 0 5.04 4.9 ± 0 . 3 6.07 7.1 ± 1.0 9.75 10.1 ± 1.2 12.1 ± 0 . 7 —
_
In Veterinary Drug Residues; Moats, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
Percent RSD — 6.0 15 12 5.9
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15.
LOPEZ-AVTIA & BENEDICTO
Veterinary Drugs in Dry Milk Powder
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SFE Procedure. The SFE extractions were performed with an Isco SFX 3560 (Isco, Inc., Lincoln, NE) automated extractor (24 vessels) at 450 atm and 80°C for 30 min in the dynamic mode. The flow rate of the carbon dioxide (SFE/SFC grade carbon dioxide, Air Products, Allentown, PA) modified with 10 percent methanol was approximately 1.5 mL/min. This flow was maintained using a stainless steel coaxially heated capillary restrictor (temperature 80°C). The extracted material was collected in methanol (1 mL initial volume with 0.5 mL additional volume added at 10 min intervals during extraction). After extraction, the methanol extracts were concentrated to dryness, and the extract residue was redissolved in 1 mL phosphate buffer immediately prior to ELISA. All SFE experiments were performed with 2-g portions of the powdered milk, which was spiked with sulfamethazine at 2.5 or 5 ng/g. The spike was added to the powdered milk in 50 or 100 /zL of 100-ng/mL solution of sulfamethazine in methanol. ELISA Procedure. For ELISA, 250 μL of the dry milk powder extract and 250 of the tracer solution were added to each reaction tube and were incubated for 3 to 4 min at room temperature (the tubes were kept on a mechanical shaker during the incubation period). Following incubation, the reaction tubes were washed at least 6 times with the wash solution (saline and surfactant) provided with the Idetek kit. Solution 2 (color developer, 500 μL) was then added to each tube and the tubes incubated for an additional 3 to 4 min. The reaction was terminated by the addition of 500 of the stopping solution (sodium dodecyl sulfate). Spectrophotometric analysis of the final colored product was performed using the Ohmicron RPA-1 photometric analyzer (Ohmicron Corporation, Newtown, PA) set at 405 nm. The observed sample results were compared to a linear regression line using a log-logit standard curve prepared from analysis of calibration standards at 0, 5, 7.5, and 10 ng/mL. An additional standard at 15 ng/mL was evaluated for calibration; however, it was found to exceed the linear range of the assay. Results and Discussion Preliminary experiments conducted with supercritical carbon dioxide at 350 atm/60°C and 450 atm/80°C using a 30-min extraction time indicated that sulfamethazine could not be extracted from the dry milk powder when spiked at 5 ng/g. Extraction with supercritical carbon dioxide at 450 atm/120°C resulted in a high background and, thus, inconclusive results by ELISA. Use of carbon dioxide modified with 10 percent methanol resulted in quantitative extraction (average recovery of 91.7 ± 17 percent) of sulfamethazine from dry milk powder spiked at 5 ng/g (Table I). Additional experiments performed with modified supercritical carbon dioxide at lower pressures (e.g., 350 atm and 250 atm) indicated poor recovery of sulfamethazine when spiked at 5 ng/g. Furthermore, addition of triethylamine (TEA), as a matrix modifier, had no effect upon recovery when 20 μL of neat TEA were added, but lowered the recovery to 68 percent when 40 μL of neat TEA were used in the extraction; therefore, we concluded that there was no need to use a matrix modifier. The linearity of the ELISA was verified with standards at 0, 5, 7.5, 10, and 15 ng/mL. As shown in Table II, the 15-ng/mL standard exceeded the linear range.
In Veterinary Drug Residues; Moats, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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The calibration standards were analyzed over a period of two weeks; the percent RSDs shown in Table II indicate that method precision is within 15 percent. The data presented in Table III indicate no background interferences at 1 ng/g in the method blank and acceptable recoveries for the blowdown evaporation step. Also shown in Table III is method performance for sulfamethazine spiked into dry milk powder at 2.5 ng/g. Based on these data, we concluded that sulfamethazine can be recovered from dry milk powder by supercritical fluid extraction and that analysis by ELISA allows detection of sulfamethazine concentrations as low as 2.5 ng/g. Work in progress in our laboratory is addressing other veterinary drugs including oxytetracycline and penicillin G. Preliminary experiments indicate that the former compound cannot be extracted from the spiked dry milk powder by SFE with supercritical carbon dioxide alone or supercritical carbon dioxide modified with methanol using in-situ derivatization with trifluoroacetic anhydride. The latter compound is a β-lactam and is not stable in methanol; therefore, another modifier is being investigated for this purpose. Table m. Verification of SFE/ELISA Procedure Step
Percent recovery
Method blank
ND (detection limit 1 ng/g)
Methanol spiked at 10 ppb, blowdown evaporation, and reconstitution in buffer
96.1 (n=2)
Nonfat powdered milk spiked at 2.5 ng/g
63.7 ± 2.8 (n=3)
Acknowledgment The authors wish to thank Les Myers of Isco, Inc., for making the Isco SFE-3560 available for use in this study, and Idetek Corporation for donating the ELISA test kits. Literature Cited 1.
Malik, S., Duncan, S. E., Bishop, J. R., and Taylor, L. T., J. Dairy Sci. 1994 77, 418.
2.
Cross, F. R., Ezzell, J. L., and Richter, Β. E., J. Chromatogr.Sci.1993 31, 162.
3.
Parks, O. W., and Maxwell, R. J., J. Chromatogr.Sci.1994 32, 290.
4.
Tena, M . T., Luque de Castro, M . D., and Valcarcel, M . , Chromatographia 1995 40, 197.
5.
Lopez-Avila, V., Charan, C., and Beckert, W. F., TRAC 1994 13, 118.
In Veterinary Drug Residues; Moats, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
