Liquid chromatographic determination of carbadox and desoxycarbadox in medicated feeds and in porcine gastrointestinal tract

A liquid chromatographic method for the assay of carbadox and desoxycarbadox in medicated feeds and porcine stomach and intestinal contents is described. Samples were extracted with dimethylformamide-water and cleaned up on an alumina column. The eluate was chromatographed using either gradient elution for simultaneous assay of both compounds or isocratic elution for carbadox only. Detection of carbadox by its native fluorescence yielded a sensitive and specific assay without interferences by metabolites or matrix components. The optimal UV absorption of desoxycarbadox was at 280 nm. Mean recoveries of carbadox in spiked feed and stomach contents were 104 and 97%, respectively; mean recovery of desoxycarbadox in stomach contents was 106%. The day-to-day reproducibility for carbadox in different feed samples and stomach contents samples had a coefficient of variation of 6-13%.     

Liquid chromatographic determination of carbadox residues in animal feed

A liquid chromatographic (LC) method for determining residues of carbadox in the 0.01-10 ppm range in swine feed is described. Carbadox is extracted from ground feed with 25% acidified methanol-CHCl3, removed from emulsion-forming coextractables via an alumina column, separated from highly colored pigments by acid-base liquid-liquid partitioning, and finally isolated from interferences on a second alumina column. Isocratic reverse phase LC at 305 nm is used for quantitation. The average overall recovery at the 0.1, 0.5, and 1.0 ppm spike levels was 83.0% with a standard deviation of 2.04% and a coefficient of variation of 2.46%.     

Modified method for carbadox in feeds: collaborative study

The official first action method for carbadox in swine feed, 42.C01-42.C04, was modified in 2 respects. First, the samples were leached overnight at room temperature instead of boiled for 1 hr. This change avoided problems with overheating and excessive evaporation. Second, the dilution scheme for samples spiked with carbadox standard solution was changed to give absorbance values that were within the optimum working range of all types of spectrophotometers. The modified procedure was collaboratively studied by 21 laboratories. The repeatability standard deviation (sigma0) and reproducibility standard deviation (sigmax) were sigma0 = 0.00029% and sigmax = 0.00056% (8.9% of grand mean) for feeds containing 0.00617% carbadox; and sigma = 0.0012% and sigmax = 0.0019% (9.3% of grand mean) for feeds containing 0.0198% carbadox. The between-laboratory variance ratio was significant for feeds containing 0.0198% carbadox. The mean per cent of intent values for feeds containing 0.00617% carbadox and 0.0198% carbadox were 102% and 104%, respectively. In general, the statistical results were comparable to those previously obtained for the official first action method. Consequently, the modified procedure is not recommended as a replacement for the official first action method.     

New microbiological method for determining spectinomycin in pelleted and meal feeds using trifluoroacetic acid as primary extractant

A new microbiological method, identified as the spectinomycin trifluoroacetic (SPE-TFA) method, was compared with the current AOAC method for analyzing spectinomycin in meal and pelleted feeds fortified with LS-20 premix. Feeds containing 3 concentrations of drugs and a zero level were tested in a correlation study. The data showed no significant differences in the percent of theory assayed between meal and pelleted samples using the SPE-TFA method, but the percent of theory found using the AOAC method was significantly lower for the pelleted samples than for the meal samples. The within-sample variation of the AOAC assay was also not the same for all samples; the SPE-TFA assay variation was relatively constant for all samples. The SPE-TFA method produced an overall average recovery of 98% with a range of 89-109% compared with an 85% recovery ranging from 64 to 102% for the AOAC method. In addition to producing better recoveries, the SPE-TFA method features a more sensitive response line, and final test solutions have viscosities and clarity more comparable to the standard solutions than those produced by the current AOAC method.     

Soyasaponins resist extrusion cooking and are not degraded during gut passage in Atlantic salmon (Salmo salar L.)

The stability of soyasaponins in fish feed formulations was investigated. The level of soyasaponin Ab, Bb, Bc, Ba-2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (Ba-DDMP), Bb-DDMP, and Bc-DDMP was quantified in 15 samples of defatted soybean meal, two full fat soybean meals, and two soybean protein concentrates by reverse phase high-performance liquid chromatography. The total level of saponins in the 15 samples of commercial defatted soybean meal ranged from 4.8-6.8 micromol/g (5.1-7.0 g/kg). The two full fat meals contained 4.4 and 4.7 micromol/g whereas no saponins could be detected in the alcohol-extracted soybean protein concentrates. Fifteen batches of fish feed containing 20% defatted soybean meal were produced by twin-screw extrusion from the 15 different samples of defatted soybean meal. Extrusion did not reduce the total level of group B saponins, but the ratio between DDMP-conjugated group B saponins and non-DDMP-conjugated group B saponins was slightly reduced. A soybean-containing diet was fed to seawater adapted Atlantic salmon for 9 weeks. Yttrium oxide was included in the feed as an inert marker in order to estimate the disappearance of saponins during gut passage. High levels of intact non-DDMP-conjugated group B soyasaponins were found in feces whereas only low levels of DDMP-conjugated saponins could be detected. The overall disappearance of saponins was close to zero, and the concentration of intact saponins in dry feces reached levels several fold higher than dietary levels. The present work demonstrates that non-DDMP-conjugated group B soyasaponins resist extrusion cooking and remain intact during gut passage in Atlantic salmon. The latter is contrary to earlier findings in endothermic animals.     

Liquid chromatographic determination of lasalocid sodium in chicken skin: interlaboratory study

The U.S. Food and Drug Administration (FDA) sponsored an interlaboratory study of a liquid chromatographic determinative procedure for lasalocid sodium in chicken skin with adhering fat. Four laboratories analyzed 35 dosed tissue samples and 82 fortified tissue samples containing lasalocid at levels ranging from 0.1 to 0.6 ppm. Samples were homogenized with acetonitrile, washed with hexane, and partitioned into the mobile phase prior to analysis liquid chromatography with fluorescence detection. The results of the interlaboratory study showed good reproducibility for fortified samples. Fortification levels, average recoveries, and interlaboratory percent coefficients of variation were as follows: 0.6 ppm, 0.57 ppm, and 9.7; 0.3 ppm, 0.25 ppm, and 9.1; and 0.15 ppm, 0.14 ppm, and 7.0, respectively. Data for analysis of the dosed tissue also showed good agreement among the laboratories.     

Liquid chromatographic determination of chloramphenicol residues in meat: interlaboratory study

A liquid chromatographic (LC) method for the determination of chloramphenicol (CAP) residues in meat at the 10 microgram/kg level was tested in an interlaboratory study. The method used, based on aqueous extraction and sample cleanup with a cartridge containing Extrelut, was published earlier. A prestudy to familiarize collaborators with the method was performed before the actual interlaboratory precision study. The meat samples used in the precision study were prepared by diluting dosed chicken and pig muscle tissues with blank tissues from other species. Fourteen laboratories received 20 meat samples; 13 laboratories actually participated in the study. Two blank samples and 2 positive samples each of pig, calf, chicken, lamb, and cow meat were tested. The chloramphenicol concentrations in the positive samples ranged from 6.5 to 21 micrograms/kg. The overall mean reproducibility coefficient of variation was 17.9% after the results per laboratory were corrected for the mean recovery obtained within each sample series. The overall mean recovery was 55.1% with a coefficient of variation of 18.0% at the 10 micrograms/kg level. The limit of detection, based on chromatograms of blank samples, was estimated to be 1.5 micrograms/kg of chloramphenicol. No false positives or false negatives were observed in the concentration range tested; only 2 false positive results above the detection limit (1.7 and 6 micrograms/kg) on a total number of 60 blank analyses (3.3%) were observed.     

Liquid chromatographic determination of carbadox, desoxycarbadox, and nitrofurazones in pork tissues

A liquid chromatographic (LC) method has been developed for the determination of carbadox, desoxycarbadox, and nitrofurazones in the 10-40 ppb range in pork muscle, liver, and kidney tissues. Tissues were homogenized in absolute ethanol, and the homogenates were treated with metaphosphoric acid and reduced in volume by rotovaporization. Hexane was added to the concentrates, which were then centrifuged to remove fat. After addition of KH2PO4 to the aqueous phase and extraction with ethyl acetate, the extracts were passed through alumina columns before analysis by reverse phase LC. Overall average recoveries (10-40 ppb range) for carbadox and desoxycarbadox from spiked tissues were 53% +/- 13.6 and 61% +/- 7.2, respectively; overall average recoveries for nitrofurazone and furazolidone were 43% +/- 7.3 and 77% +/- 10.9, respectively. Before these optimum determinations, degradation by even minimal incandescent light was found to reduce recovery especially of desoxycarbadox. The results of this photochemical degradation are reported and briefly discussed.     

Interlaboratory comparison of analyses for heavy metals in clam tissue

Results are reported for an interlaboratory study conducted to assess the reproducibility of analyses for lead, copper, cadmium, and zinc. The 10 participating laboratories analyzed 2 samples of freeze-dried clam tissue and 1 disguised sample of NBS Oyster Tissue. Interlaboratory variations were observed for all metals, although the methods yielded reproducible data for Cu and Zn with average interlaboratory coefficients of variation of 15 and 11%, respectively. The performance of methods used for Cd and, more so, for Pb was less than satisfactory. Cadmium levels in the 3 samples ranged from about 0.7 to 3.7 ppm with an average interlaboratory coefficient of variation (CV) of 24%. Lead levels in the 3 samples were about 0.5 ppm with an interlaboratory CV of 68%. Some laboratories' results were consistently high or low but data were insufficient to relate these trends to one particular variable. Results from this study were compared with 5 other studies reported in the literature since 1980. Coefficients of variation from all studies were comparable for samples with similar metal concentrations.     

Determination of sulfamethazine and trimethoprim in liquid feed premixes by HPLC and diode array detection, with an analysis of the uncertainty of the analytical results.

Sulfamethazine (SMZ) and trimethoprim (TMP) are antibacterials used in veterinary practice. This paper describes a method for their determination in veterinary liquid feed premixes that is based on liquid chromatography with diode array detection. Gradient elution with methanol and ammonium acetate achieved excellent separation of the two analytes within 15 min without any interference from the matrix. Absorbance of the column effluent was monitored at 264 nm for SMZ and at 230 nm for TMP. Detailed analyses of the uncertainties of determinations afford estimated expanded uncertainties of, respectively, 0.2 and 0.1 w/v % for typical SMZ and TMP concentrations of 10.7 and 2.1 w/v %, respectively. At the lower end of the calibrated range of the method, the dominant source of uncertainty is the preparation of standards and the construction of the calibration line.     

Liquid chromatographic method for determination of furazolidone in premixes and complete feeds: collaborative study

A liquid chromatographic (LC) method for determining furazolidone in finished feeds and premixes was collaboratively studied. Finished feed sample is extracted with acetone-water (93 + 7) on a Goldfisch apparatus, extracting solvent is removed, and the residual material is dissolved in warm DMF. A solution of tetraethylammonium bromide is added, the fat layer is removed, and the sample is clarified by filtration and injected onto a reverse phase LC system with detection at 365 nm. Premixes, extracted by shaking with DMF and diluted so that the final furazolidone concentration is about 55 micrograms/mL, are chromatographed and detected the same as finished feed samples, using a mobile phase of acetonitrile-2% acetic acid (20 + 80). Ten commercial feed samples were preweighed and supplied to 14 collaborators. The 5 matched pairs were chosen to represent the following allowed levels: 0.0055, 0.022, 0.033, 2.2, and 22%. Two familiarization samples at the 0.0055 and 11% levels were also supplied. Instructions called for a single analysis of each sample. Two results were eliminated by the Dixon test. The coefficients of variation, following treatment by the ranking test, ranged from 2.0 at the 22% level to 6.5 at the 0.0055% level. Calculated F-values are not significant (P greater than 0.01) except for the 0.0055% level samples extracted overnight. This method has been adopted official first action.     

Liquid chromatographic determination of morantel tartrate in cattle feed

A liquid chromatographic (LC) method is proposed for measuring 0.485-0.970% morantel tartrate in cattle feeds. The drug is leached from feed, diluted, separated from interfering substances on a silica column, and measured in the effluent stream by 313 nm spectrophotometric detection. Two potential degradation products, i.e., cis-isomer of morantel tartrate and N-(3-methylaminopropyl)-trans-3-(3-methyl-2-thienyl)acrylamide, and a related anthelmintic, i.e., pyrantel tartrate, do not interfere. Average recovery of drug from liquid spiked samples and laboratory blends was 98-100% with a maximum coefficient of variation (CV) of 2.3%. Results for pelleted and crumbled commercial scale feeds ranged from 94 to 102% of label claim, with a maximum CV of 1.5%.     

Liquid chromatographic determination of amprolium in poultry feed and premixes using postcolumn chemistry with fluorometric detection

Two extraction and liquid chromatographic procedures are presented which separate amprolium from compounds in poultry feed or premixes that could interfere with its fluorometric determination. The procedures are based on earlier work on the determination of thiamine in food samples. Amprolium is extracted from feed with a hexane-aqueous sulfosalicylic acid mix, separated on a C18 column, and detected fluorometrically after postcolumn derivatization. For premixes, water extraction is used. Values for the amprolium content of poultry feed obtained with these procedures are in good agreement with those obtained with AOAC official methods. It is suggested that these methods with suitable modifications may be of use for routine analysis of amprolium in feeds. The overall methods are rapid and appear to give reasonable results.     

Effects of grinding and storage for one month on retention of vitamin A in premixes and mineral supplements

Tested standardized procedures for handling premixes and mineral supplements from time of sampling to time of analysis for vitamin A have not been developed, which could account for some unexplained inconsistent and low analytical results. Grinding premix samples and storing them in a freezer for one month had little effect on amount of vitamin A found, but there was a significant loss (about 10%) after storage for one month at room temperature. Results on replicated determinations of vitamin A in unground and ground mineral supplements and on effect of storage were somewhat more variable than for premixes, but only the loss (about 12%) during storage for one month at room temperature was significant.     

Nutritional and Economic Implications of Protein Variance and Application of Statistical Process Control in the Chinese Feed Industry

The adoption of Statistical Process Control (SPC) in the Chinese feed industry to reduce finished feed protein and moisture content variation can improve feed quality and profitability. Two studies were conducted to characterize protein variation in the Chinese feed industry. Study I involved four participating feed mills where we collected three samples per bag from four bags per lot of soybean meal, rapeseed meal, fish meal, corn gluten meal, meat & bone meal, and cottonseed meal. We analyzed these ingredients for protein and moisture content. Cottonseed meal and meat & bone meal displayed the highest protein variability between bags and within bags compared with other protein ingredients. We found no significant difference for within‐bag protein variance of the other feed ingredients; however, soybean meal had a significantly lower between‐bag variance (0.04%). Study II assessed the effect of adopting SPC. Five feed mills provided data for one year of finished feed protein and moisture content before and after adopting SPC. Three of the five feed mills experienced a significant (P < 0.10) reduction (0.20, 0.52, and 0.70%) in the absolute finished feed protein variation after adopting SPC. The two feed mills that did not display a significant reduction in absolute protein variation were already manufacturing feed with a low (<0.4% protein) absolute deviation from the target. These data demonstrate that feed mill managers can control finished feed variability through a number of management techniques including SPC.     

Liquid chromatographic monitoring of the depletion of carbadox and its metabolite desoxycarbadox in swine tissues

A liquid chromatographic method was used to monitor a depletion study of carbadox (and its most important metabolite, desoxycarbadox) in young pigs fed carbadox-treated rations for 1 week. Carbadox was found in blood (20 ppb), blood serum (26 ppb), and muscle tissue 24 h after withdrawal from treated ration; residues were reduced to a trace (less than 2 ppb) in 48 h, and eliminated by 72 h. Desoxycarbadox, although not detected in blood, was found in muscle (17 ppb) 24 h after withdrawal; it was reduced to 9 ppb at 48 h and to a trace by 72 h. Although no carbadox was detected in liver 24 h after withdrawal, appreciable desoxycarbadox (125 ppb) was found in liver 24 h after withdrawal; it was reduced to 17 ppb at 48 h and to a trace by 72 h. Whereas only a trace of carbadox was found in kidney 24 h after withdrawal, 186 ppb desoxycarbadox was found in kidney at 24 h, 34 ppb at 48 h, and a trace at 72 h. No metabolite of carbadox other than desoxycarbadox was found in extracts of swine tissues during this medicated feed trial, and no metabolite was found in blood extracts by using the established methodology. The effect of tissue storage (aging) at -20 degrees C on levels of the drug and its metabolite was a modest alteration of residue levels. The inadvertent use of feed adulterated with furazolidone and initially medicated with chlortetracycline, sulfamethazine, and penicillin G, did not affect the uptake of carbadox in this depletion study or interfere with the analytical methodology.     

Liquid chromatographic determination of narasin in feed premixes

A liquid chromatographic (LC) method has been developed to determine narasin in feed premixes. Narasin is extracted from the premix with a methanol-water solvent, and the extracted solution is assayed by using LC. Recovery of narasin from a 12.5 g/lb premix is quantitative (100%), with a relative standard deviation of 1.44%. The results correlated well (coefficient 0.92) with a turbimetric bioassay method.     

Pyridine extraction method for determination of bacitracin in feeds: collaborative evaluation.

Seventeen laboratories evaluated the pyridine extraction method and neomycin-sensitized agar for the determination of zinc and MD bacitracin in swine and broiler rations at 10 and 100 g/ton. The method was also applied to the analysis of 2 premixes labeled 50 g/lb (MD bacitracin) and 40 g/lb (zinc bacitracin). Bacitracin activity was determined on each of 2 days with 2 dilutions on each day. No significant difference was found between dilutions within a day or between days for each sample. The type of bacitracin or type of feed did not significantly affect results. The difference in results between MD and zinc bacitracin in premixes approached significance. The large coefficients of variation for premixes (ca 13%) and complete feeds (ca 15--30%) indicate operational problems. The main difficulty was evaporation of pyridine. Some laboratories were not able to evaporate it completely, whereas others lost bacitracin activity, probably due to high temperature of drying. The pyridine extraction method as in 42.200 and 42.204 should be discontinued.     

Liquid chromatographic determination of saccharin in beverages and sweets: NMKL Collaborative Study

A reverse phase liquid chromatographic method for the determination of saccharin in a soft drink and a juice was collaboratively studied in 8 laboratories. Collaborators were supplied with 3 samples of the soft drink and 3 samples of the juice containing sodium saccharin levels of 40-100 mg/L. Average recoveries of sodium saccharin were 95.3% for the soft drink and 98.0% for the juice. The reproducibility coefficients of variation were 16.9% for the soft drink and 10.4% for the juice. In addition, a mini-collaborative study was conducted for the determination of saccharin in 3 samples of sweets produced commercially. Five collaborators analyzed the samples, which contained saccharin at levels of 100-600 mg/kg according to the maker's specifications. Saccharin was extracted with water and ethanol and chromatographed using a modified liquid chromatographic method. The reproducibility coefficient of variation was 12.4% for the sweets.     

Detection of low-level carbadox residues in animal feeds by high pressure liquid chromatography

The high pressure liquid chromatographic (HPLC) method is capable of detecting from 1 to 0.024 ppm methyl 3-(2-quinoxalinyl-methylene)carbazate-N1,N4-dioxide (carbadox). Carbadox is extracted from the feed with 2% NH4OH in acetone, passed through a liquid-liquid partition, subjected to HPLC, and detected by using a 365 nm detector. No feed materials or other active drug ingredients produced false positive results.