Anal. Chem. 1998, 70, 2415-2420
Technical Notes
Assay for Cephapirin and Ampicillin in Raw Milk by High-Performance Liquid Chromatography-Integrated Pulsed Amperometric Detection Catherine O. Dasenbrock and William R. LaCourse*
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250
The FDA has issued guidelines governing the use of antibiotics in cattle and routinely tests for the presence of antibiotics in milk. Unfortunately, these compounds are often difficult to detect by direct methods because they often lack a chromophore or fluorophore. Integrated pulsed amperometric detection (IPAD) following reversedphase liquid chromatography is well-suited for this analysis because it is selective, sensitive, and direct; i.e., derivatization is not required. This work involves the development of a simple, rapid assay for the determination of β-lactam antibiotic residues in milk using HPLCIPAD, specifically, ampicillin and cephapirin. Since the analytes studied here are detectable by UV detection, a comparison between IPAD and UV detection will be made. Sample preparation schemes that involve the extraction of antibiotics of interest from the milk matrix and subsequent cleanup are an important aspect of this project. These procedures will be discussed in detail. In addition, analytical figures of merit and IPAD wave form optimization will be addressed. Detection of antibiotics in food is becoming increasingly important. The ubiquitous presence of antibiotics in food can have serious health consequences for consumers that range from allergic reactions to the evolution of antibiotic-resistant bacteria, which may eventually lead to the need for stronger antibiotics. Dairy cows are routinely dosed with antibiotics to fight infections, such as mastitis, and to maintain good health.1 For these reasons, the presence of antibiotics in milk is regulated by the Food and Drug Administration (FDA). Detection of antibiotics at trace levels is not trivial in many cases, as many antibiotics of interest have a weak chromophore. In addition, biological fluids are challenging matrixes. Current extraction techniques for antibiotics from milk can be difficult to perform, require the use of toxic solvents, or not be adequately sensitive for the determination. Reports of extractions (1) Bishop, J. R.; Bodine, A. B.; O’Dell, G. D.; Janzen, J. J. J. Dairy Sci. 1984, 67, 437-440. S0003-2700(97)01375-9 CCC: $15.00 Published on Web 05/01/1998
© 1998 American Chemical Society
of cephapirin from milk2 and β-lactam antibiotics from milk3-9 and tissues10,11 have been published. Many of these protocols rely on low-wavelength UV detection for the liquid chromatographic analysis step and therefore require difficult sample preparation protocols5,9,11-13 or postcolumn derivatization7,14 in order to obtain adequate sensitivity to accomplish the analysis. Pulsed electrochemical detection (PED) has been shown to be effective for the detection of sulfur-containing compounds following HPLC.15-17 Several different techniques are grouped under the heading of PED, which denotes a detection step (e.g., amperometric, coulometric) that is coupled with pulsed potential cleaning and reactivation steps. Sulfur-containing compounds are detected via an oxide-catalyzed mechanism; oxide formation at the electrode surface occurs simultaneously and is required for the detection process. Unfortunately, formation of the gold oxide is associated with unstable baselines and large background signals. To alleviate these problems, a linear, cyclic scan between two potentials is performed during the detection step.17-20 This detection scheme is called integrated pulsed amperometric detection (IPAD). The potential scanning allows the charge from oxide (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20)
MacIntosh, A. I. JAOAC Int. 1990, 73, 880-882. Kirchmann, E.; Welch, L. E. J. Chromatogr. 1993, 633, 111. Terada, H.; Sakabe, Y. J. Chromatogr. 1985, 348, 379-387. Fletouris, D. J.; Psomas, J. E.; Mantis, A. J. J. Agric. Food Chem. 1992, 40, 617-621. Moats, W. A. J. Chromatogr. 1990, 507, 177-185. Rogers, M. E.; Adlark, M. W.; Saunders, G.; Holt, G. J. Chromatogr. 1983, 257, 91-100. Boison, J. O. K.; Keng, L. J.-Y.; MacNeil, J. D. JAOAC Int. 1994, 77, 565570. Moats, W. A.; Harik-Khan, R. JAOAC Int. 1995, 78 (1), 49-54. Boison, J.; Keng, L. Semin. Food Anal. 1996, 1, 27-32. Moats, W. A. J. Chromatogr. 1984, 317, 311-318. Terada, H.; Sakabe, Y. J. Chromatogr. 1985, 348, 379-387. Tarbin, J. A.; Farrington, W. H. H.; Shearer, G. Anal. Chim. Acta 1995, 318, 95-101. Boison, J.; Keng, L. Semin. Food Anal. 1996, 1, 33-38. Dasenbrock, C. O.; LaCourse, W. R. CHEMTECH 1998(1), 26-32. Vandeberg, P. J.; Johnson, D. C. Anal. Chem. 1993, 65, 2713-2718. LaCourse, W. R.; Owens, G. S. Anal. Chim. Acta 1995, 307, 301-319. Johnson, D. C.; LaCourse, W. R. Anal. Chem. 1990, 62, 589A-597A. Vandeberg, P. J.; Johnson, D. C. Anal. Chim. Acta 1994, 290, 317-327. Koprowski, L.; Kirchmann, E.; Welch, L. E. Electroanalysis 1993, 5, 473482.
Analytical Chemistry, Vol. 70, No. 11, June 1, 1998 2415
formation to be electronically subtracted from the total signal. This wave form increases baseline stability, resulting in better reproducibility for sulfur-containing compounds.19,21 In addition, IPAD at a gold electrode under mildly acidic conditions has been shown to be selective for sulfur-containing compounds.15,17 The use of PED following HPLC for the detection of sulfur-containing compounds has been reviewed.22 IPAD wave form consists of three basic steps: a detection step, an oxidation/cleaning step, and a reduction/reactivation step. During the first step, the starting detection potential (Edst) is held for a delay time (tdel) to allow for the decay of charging currents; then, the potential is scanned linearly from Edst to the maximum detection potential (Edmx) to the ending detection potential (Ednd). The entire cyclic scan is integrated over time (tint), which constitutes the analytical signal. The potential is held at Ednd for time thld. In the second step, surface oxide is formed by changing the potential to Eoxd and holding for toxd. The final step is the reduction of the surface oxide to a clean gold surface by holding Ered for tred. Some adsorption of the analyte to the gold surface also takes place during the reduction step. Although some cleaning takes place during the cyclic potential scan as with CV, additional cleaning through the formation of a fully developed oxide layer is necessary for strongly adsorbed contaminants. IPAD offers advantages for the analysis of sulfur-containing antibiotics in complex matrixes over existing detection schemes. These advantages include the ability for trace analysis, selective detection, which will yield a less complex chromatogram, and simplified sample preparation. Additionally, because IPAD is a direct technique, derivatization is not necessary. The focus of this work is the extraction of cephapirin and ampicillin from milk and analysis of the extracts by HPLC-IPADUV. They are structurally similar; they are both β-lactam antibiotics which have a UV chromophore and at least one sulfurcontaining group. Because these analytes can be detected by either IPAD or UV detection, both are used in this work to directly compare the two detection methods. EXPERIMENTAL SECTION Materials. All solvents were HPLC grade; all solutions were filtered through a 0.2-µm filter prior to use in the HPLC system. All antibiotic standards were purchased from Sigma (St. Louis, MO) and were prepared in water. All standard solutions, if not used immediately, were stored either in the refrigerator or freezer overnight. Water was purified using a reverse-osmosis system coupled with multitank/ultraviolet/ultrafiltration stations (US Filter/Ionpure, Lowell, MA). Voltammetry. Cyclic voltammetric data were collected at a gold rotating disk electrode (RDE) using a model BAS RDE-1 rotator and a BAS 100-B (Bioanalytical Systems, West Lafayette, IN) electrochemical workstation with a 75-MHz Pentium computer (Gateway 2000, North Sioux City, SD). The reference electrode was Ag/AgCl (BAS) and the auxiliary electrode was a Pt wire. Cyclic voltammograms were taken using a scan rate of 250 mV/s and a rotation speed of 900 rpm. Chromatographic System. The chromatographic system consisted of a GP-40 chromatography pump (Dionex, Sunnyvale, (21) Neuberger, G. G.; Johnson, D. C. Anal. Chem. 1988, 60, 2288-2293. (22) Johnson, D. C.; Dobberpuhl, D.; Roberts, R.; Vandeberg, P. J. Chromatogr. 1993, 640, 79-96.
2416 Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
CA) with an ED-40 electrochemical detector (Dionex) equipped with a gold working electrode, a pH reference electrode, and a titanium counter electrode. A UV detector (Dionex VDM-2.) was also used for on-line comparisons and was situated after the electrochemical detector. The separations were accomplished with a Luna C-8 analytical column (Phenomenex, Torrance, CA) 5-µm particle size, 150 × 4.6 mm, and a Symmetry C-8 guard column (Waters Corp., Milford, MA). The columns and the electrochemical cell were temperature controlled at 30 °C with an LC-30 chromatography oven (Dionex). Data were collected via computer with PeakNet software (Dionex). The data from the electrochemical and UV detection were smoothed using a Savitsky-Golay algorithm with a filter size of 11 points and two iterations. Unless otherwise specified, the mobile phase was 20% A, 20% B, and 60% C, where A is 20% acetonitrile; B is 500 mM acetate buffer, pH 3.75; and C is water. Extraction Procedure. Raw milk was obtained from the FDA-Center for Veterinary Medicine (CVM) and stored at -80 °C until use. Raw milk was fortified with cephapirin and ampicillin such that 1 mL of antibiotic solution is added to 10 mL of milk. For the blank milk, 1 mL of deionized water was added to 10 mL of milk. The milk mixture was vortex mixed for 20 s. The milk samples were then centrifuged at 5000 rpm for 30 min at room temperature. Upon removal from the centrifuge, the samples were placed on ice for 10 min to allow the top fat layer to solidify. The skim milk could then be decanted, and the fat layer was discarded. To precipitate the proteins, 20 mL of acetonitrile was added slowly with stirring. After all of the acetonitrile was added, the mixture was allowed to stir for an additional 5 min and then to stand for 5 min. The mixture was then filtered through a glass wool plug to remove the precipitated proteins. An additional 2 mL of acetonitrile was added to rinse the remaining sample through the glass wool plug. The volume of the extract was reduced to 1-2 mL by evaporation on a rotary evaporator (Buchner). A 200-mg C-18 Sep-Pack cartridge (Waters) was conditioned with 5 mL of methanol, 5 mL of water, 5 mL of 2% NaNO3, and 5 mL of 100 mM phosphate buffer pH 8.5, and then the milk extract was applied to the cartridge. The vessel in which the extract was evaporated was then rinsed with 3 mL of 100 mM phosphate buffer pH 8.5, and these washings were added to the extraction cartridge. The cartridge was then washed with 2.25 mL of 5% acetonitrile. The analytes were eluted with 0.95 mL of 15% acetonitrile, and this eluant was transferred to a 1-mL volumetric flask and diluted to the mark with water. The sample was filtered with a 0.45-µm syringe filter and injected onto the HPLC-IPAD-UV system. RESULTS AND DISCUSSION Voltammetry and Wave Form Optimization. Cyclic voltammetry was used to study the electrochemical response of the analytes because the cyclic wave form used in CV mimics the cyclic scans performed during the detection step in IPAD. Figure 1A shows a cyclic voltammogram of cephapirin in 500 mM acetate buffer pH 3.75 and the residual scan. The residual scan shows the oxide formation starting at about 1050-1350 mV. On the reverse scan, the oxide reduction is evident at 500-800 mV. When cephapirin is added, an oxidation signal is observed to occur simultaneously with the gold oxide formation. A shift in the oxide formation wave is also observed because the adsorption of
Figure 1. Cyclic voltammetry of cephapirin at a gold RDE: (A) 500 mM sodium acetate pH 3.75 and (B) and 4% acetonitrile, 500 mM sodium acetate pH 3.75. Scans: (s) 17.8 mM cephapirin; (‚‚‚) residual.
cephapirin suppresses the onset of gold oxide formation. A subsequent oxidation for cephapirin is not observed because the fully formed monolayer of gold oxide on the electrode surface blocks all other electrode reactions.20,24,25 On the reverse scan, only the signal for the reduction of the monolayer of gold oxide is observed. Figure 1B shows the response for cephapirin in 4% acetonitrile, 500 mM sodium acetate buffer pH 3.75. The acetonitrile was added because it is present in the mobile phase. A broadening of the oxidation wave is evident, and the shift in the oxide formation upon the addition of the analyte is not present. Since the oxide formation is already perturbed from the presence of the acetonitrile, this observation was not unexpected. The magnitude of the reduction wave for the surface oxide corresponding to a monolayer of gold oxide is not changed with the addition of cephapirin, as noted by the similar area of the reduction wave for both plots. Hence, background subtraction inherent in the IPAD wave form will effectively yield only the current associated with the analyte. In choosing the potentials to be used in the wave form, the following guidelines were used:15,25 i. The cyclic scan of Edet must begin (Edst) and end (Ednd) in the oxide free potential region; i.e., E < 0.700 V vs Ag/AgCl for Au in 0.5 M sodium acetate pH 3.75 (Figure 1). ii. Ednd should end before cathodic detection of dissolved O2 when working with nondeoxygenated solutions (E > 0.0 V vs Ag/ AgCl for Au in 0.5 M sodium acetate pH 3.75). iii. Edmx cannot extend into the region where anodic solvent breakdown occurs (E >1.600 V Ag/AgCl for Au in 0.5 M sodium acetate pH 3.75). From a practical standpoint, scanning up to the region of solution breakdown is undesirable because the noise is increased, reducing the S/N achievable. Figure 1 also shows the range of the potential scan using the optimized wave form. It is important to note that the detection cell utilized a pH reference electrode and the cyclic voltammetry (23) Neuberger, G. G.; Johnson, D. C. Anal. Chem. 1988, 60, 2288-2293. (24) LaCourse, W. R. Pulsed Electrochemical Detection in High Performance Liquid Chromatography; John Wiley: New York, 1997. (25) LaCourse, W. R.; Dasenbrock, C. O.; Zook, C. M. Semin. Food Anal. 1997, 2, 5.
Table 1. Optimized IPAD Wave Form Used for This Work
a
time, ms
potential,a mV
0.00 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 1.00 1.01 1.11 1.12 1.82
50 50 1150 50 1150 50 1150 50 1150 50 50 1600 1600 -400 -400
integration period begin
end
Versus pH reference.
was done versus an Ag/AgCl reference. Using the above guidelines and emperical testing, Edst and Edmx were chosen to be 240 and 1340 mV, respectively. Over this potential range, the analyte oxidation takes place. The potential was scanned between Edst and Edmx for four cycles to improve the signal-to-noise ratio in the shortest time. The Eoxd was chosen to be 1790 mV to achieve a fully formed gold oxide layer to allow for complete cleaning of the electrode surface. Ered was -210 mV; this was empirically determined to be the best Ered because the response was stable for months of continuous use. Less negative potentials were not as effective for electrode cleaning because the electrode required mechanical polishing every 1-2 months with continuous use and more negative potentials caused a decrease in the analyte response. The wave form used in this work is shown in Table 1. Reversed-Phase Chromatography. The analytes were separated by a reversed phase mechanism on a C-8 column with acetonitrile as the modifier. Isocratic elution chromatography was used throughout this project because large unacceptable baseline shifts are observed when IPAD detection is coupled with gradient elution chromatography at high sensitivity, making detection difficult.26,27 Acetonitrile was chosen for the organic modifier Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
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Table 2. Analytical Figures of Merit for Cephapirin and Ampicillin Using IPAD line equation [responsea ) m(ppb) + b] compound
detector
LOD (S/N ) 3), ppb
target level, ppb
m
b
r2
cephapirin ampicillin cephapirin
IPAD IPAD UV
5 5 20
20 10 20
0.018 303 0.008 750 2 3.211 2
0.006 531 4 0.053 800 0.029 578
0.999 39 0.999 40 0.999 15
a
For IPAD the response is in nC; for UV detection the response is in mAU.
Figure 2. Separation of a standard mixture of (1) 400 ppb cephapirin and (2) 200 ppb ampicillin. Chromatographic conditions are as described in the Experimental Section.
because it does not interfere with PED detection of sulfur containing compounds.16,17,22 A chromatogram of a standard mixture of cephapirin (400 ppb) and ampicillin (200 ppb) is shown in Figure 2. The negative peak at 8 min is associated with injection of dissolved oxygen in the sample and is always present. The capacity factors of cephapirin and ampicillin are 12.97 and 16.20, respectively. The long separation time was necessary to resolve the analyte peaks from the matrix using isocratic elution. Table 2 shows the analytical figures of merit from the calibration curves for this analysis. The limits of detection are below the target levels specified by the FDA for these compounds. In addition, there is a 10-fold concentration of the sample in the extraction protocol so the concentrations of cephapirin and ampicillin expected in the sample are well above the LOD for the method. Since we need to be able to detect the analytes at concentrations ranging from 0.5 to 2 times the target level, the requirements for the detection portion of the method are met. For comparison, a calibration curve for the UV detection at 254 nm of cephapirin was also done (see Table 2). The sensitivity for cephapirin by IPAD is 4 times that for UV detection. A calibration curve for ampicillin was not done because ampicillin was only detectable at very high concentrations, i.e., >1600 ppb. Other workers have used wavelengths ranging from 200 to 220 nm and have obtained greater sensitivity for ampicillin.9,12 How(26) Welch, L. E.; LaCourse, W. R.; Mead, D. A.; Johnson, D. C. Anal. Chem. 1989, 61, 555-559. (27) Welch, L. E.; Johnson, D. C. J. Liq. Chromatogr. 1990, 13, 1387-1409.
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ever, the background for the milk extracts at lower wavelengths (200-220 nm) was so large by our method, it precluded any detection of the analytes at these wavelengths. Extraction of Antibiotics from Raw Milk. The development of a rugged, reproducible extraction procedure for cephapirin and ampicillin from raw milk is essential to the success of this assay. To devise the best procedure, published assays were consulted. The first question encountered about the extraction was how to best accomplish removal of the milk fats. A simple procedure for fat removal from raw milk is to centrifuge at 4-5 °C until the cream has formed a layer at the top of the milk mixture.13,28 In this work, the sample is centrifuged at 5000 rpm for 30 min at room temperature and then placed in an ice/water bath. The fat layer solidifies, the skim milk is easily decanted, and the cream is discarded. There was some concern that the analyte may partition into the fat layer, making washing the cream necessary. To determine whether this step was necessary, blank raw milk (10 mL) was centrifuged and the fat layer was retrieved. Twenty milliliters of phosphate buffer pH 8.5 with 50 ppb cephapirin was then added to the fat layer, and the mixture was vortexed for 1 min. The mixture was then centrifuged for 30 min at 5000 rpm, and the resulting solution was injected onto the HPLC-UV system and compared with a 50 ppb 100 mM phosphate pH 8.5 sample. The mobile phase was 80% A (20% acetonitrile) and 20% B (500 mM acetate pH 3.75). These chromatograms are shown in Figure 3. In both cases, the peak heights for the cephapirin peak are equivalent. After the cream equilibration, the cephapirin peak is 100% of that for the standard. This result indicates that there is not significant sample loss from partitioning into the fat layer; thus washing the fat layer is not necessary. The proteins in the sample were the next concern. From published procedures, two distinct methods of protein removal are most often used. Moats and co-workers6,9 precipitate the proteins with acetonitrile and filters the remaining solution. This solution is reduced by evaporation, and the remaining solution is subjected to further cleanup. Other workers2,12 use the solid-phase extraction (SPE) step to remove the proteins by adding the milk sample directly to the SPE cartridge. Most of the proteins will pass through the cartridge. The sample is eluted then from the SPE cartridge with 100% acetonitrile because the acetonitrile will precipitate any remaining proteins onto the cartridge and they will be left behind. For this work, both procedures were tried. The former procedure did not result in a sufficiently clean sample. The latter procedure proved quite difficult because the cartridge would often become blocked, the extracts were quite dirty, and the extraction results were not reproducible. In this work, the (28) Schermerhorn, P. G.; Chu, P. S.; Kijak, P. J. J. Agric. Food Chem. 1995, 43, 2122-2125.
Figure 3. Analysis of the cream for cephapirin using UV detection: (A) 50 ppb cephapirin standard in pH 8.5 phosphate buffer; (B) 50 ppb cephapirin solution which has equilibrated with the blank cream. The mobile phase was 80% acetonitrile, 20% 500 mM sodium acetate pH 3.75.
Figure 5. Comparison of IPAD and UV detection for analysis of a milk extract: (A) IPAD; (B) UV detection. Chromatogram C is a blank extract with UV detection. Chromatographic conditions are as specified in the Expermental Section. Peak identies are (1) cephapirin and (2) ampicillin.
Figure 4. Chromatograms of (A) a milk extract from a sample fortified with (1) 40 ppb cephapirin and (2) 20 ppb ampicillin and (B) a blank milk extract. Chromatographic conditions are as discussed in the Experimental Section. Peak identies are (1) cephapirin and (2) ampicillin.
proteins are precipitated with acetonitrile and filtered with glass wool and then the sample is evaporated to 1-2 mL. The SPE step is then performed on the resulting sample. In this case, an acetonitrile/water solution can be used to elute the sample because there is no concern that proteins will interfere with the extraction. This procedure results in a much cleaner extract. During the SPE step, Terada and Sakabe12 and Boison and Keng14 condition the cartridge with a 2% NaCl solution after conditioning
with methanol and water. This was added to the procedure except NaNO3 was substituted for NaCl. Generally, Cl- is avoided when a gold electrode is used to avoid solubilization of the gold electrode. Following this step, Boison and Keng conditioned the cartridge with 100 mM phosphate buffer pH 8.5 and then added the analyte. Adding these two conditioning steps to the protocol presented here increased the extraction efficiency and the overall recovery of the extraction. Figure 4A shows the chromatogram for a milk extract for 40 ppb cephapirin and 20 ppb ampicillin; the blank extract is shown in Figure 4B. Cephapirin and ampicillin elute within 35 min and are effectively free from interferences under these conditions. There is a small peak in the blank that elutes just after the cephapirin, but it did not significantly interfere with the quantitation of the cephapirin peak. A comparison between IPAD and UV detection for the target analytes is depicted in Figure 5. Again, the cephapirin and ampicillin peaks are clearly seen using IPAD detection. The chromatogram obtained using UV detection shows a peak corresponding to cephapirin; however, there is an interfering peak which also elutes at that retention time. For comparison of the two situations, a sample was fortified with 40 ppb cephapirin; the matrix peak, which elutes just after the cephapirin using IPAD, is 10% of the analyte peak height. The interferent observed for cephapirin for UV detection is 40% of the analyte peak height. Chromatogram C shows a blank extract with UV detection and this interferent is visible. The interfering peak is sufficiently large so as to overwhelm the analyte peak at lower concentrations of Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
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Table 3. Extraction Efficiencies for Cephapirin and Ampicillin from Raw Milk compound
concn, ppb
extractn effic, %
av extractn effic, %
cephapirin
40 20 10 20 10 5
79, 78, 84, 81, 60, 64 82, 76, 80, 81, 79 78, 58, 77, 67, 85, 99 51, 76, 79, 80, 66, 48 87, 61, 92, 57, 93 84, 83, 51, 72, 93, 66
74 ( 9 80 ( 2 77 ( 13 67 ( 13 78 ( 16 75 ( 14
ampicillin
cephapirin. Ampicillin is impossibile to detect because there is a large interferent eluting at the expected retention time for ampicillin. Table 3 shows the extraction efficiencies for the milk extracts at the three levels tested. The concentrations chosen for this study were 2 times the target level, the target level, and 0.5 times the target level. The average extraction efficiency levels were greater than 70% for cephapirin and 65% for ampicillin. This was sufficient to quantitate the analytes of interest. CONCLUSIONS The method presented here for the extraction and analysis for cephapirin and ampicillin in raw milk is rugged and reproduc-
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ible and achieves the required sensitivity. The use of IPAD detection has allowed for the direct detection of the analytes at low levels and for selective detection of sulfur-containing compounds within the milk matrix, reducing the amount of sample cleanup necessary. By the method described here, UV detection at 254 nm was not sufficient with the level of sample preparation used because either there were interfering peaks eluting concurrent with the peak of interest or the sensitivity was not adequate. This method is applicable to most antibiotics in this class with minor changes to the elution protocol from the SPE cartridge and in the chromatographic conditions. ACKNOWLEDGMENT The authors thank Christine M. Zook for her contributions to this work, Dr. E. Ian DeVeau and Dr. Pak-Sin Chu for helpful discussions, Aristotle G. Kalivretenos for the use of the rotary evaporator and centrifuge, and the FDA-CVM for the donation of raw milk used in this work. The authors also acknowledge financial support from FDA Grant FD-R-000903. Received for review December 29, 1997. Accepted March 19, 1998. AC971375E
