Ivermectin pharmacokinetics in lactating sheep.

Ivermectin (IVM) concentrations in plasma and milk were studied in six Istrian Pramenka dairy sheep after a single subcutaneous dose of 0.2 mg/kg b.w. of IVM in the early lactation period to describe IVM disposition in milk and to evaluate the transfer of IVM residues via milk to suckling lambs. Large inter-animal in concentration variability of IVM in both matrices was observed. The highest overall concentration was found in the same animal: 21.7 microg/l of H(2)B(1a) in plasma on the second day and 44.9 microg/kg of H(2)B(1a) in milk on the first day after the drug was administered. The mean time in which IVM concentrations fell below the limit of detection for the whole ewe group was 22 and 23 days for plasma and milk, respectively. Time course of IVM concentration in milk was following the time course of IVM concentration in plasma, with an overall mean+/-S.D. of milk/plasma ratio of 1.67+/-0.50 for the first 7 days of the experiment. A mean of 0.7% of the dose was excreted through milk. Individual pharmacokinetic parameters were determined by fitting a one-compartment model to the milk and plasma concentration-time profiles. Mean t(max), c(max), t(1/2k(e)) and AUC values for plasma data were: 1.70+/-0.65 days, 11.88+/-6.96 microg/l, 2.85+/-1.97 days and 63.99+/-28.34 microg day/l, respectively, and for milk: 1.28+/-1.07 days, 22.67+/-18.27 microg/l, 3.56+/-2.01 days and 114.60+/-60.41 microg day/l, respectively. The highest level of concentration in suckling lamb plasma, 0.36 microg/l of H(2)B(1a), was slightly above the limit of determination. The mean lamb to ewe ratio of areas under the plasma concentration-time curve for the first 5 days was 0.02. On the basis of obtained results, it can therefore be claimed that indirect IVM exposure of the suckling lambs via milk was negligible.

Development and validation of a method for the determination of sub-additive levels of virginiamycin in compound animal feeds by liquid chromatography.

A method for the detection of virginiamycin M1 as a marker compound of virginiamycin at sub-additive level in pig, calf, piglet, sow, poultry, cattle and laying hen feeds was developed and validated. Both UV detection at 230 nm and MS detection were applied. Virginiamycin M1 was extracted from animal feeds with ethyl acetate after wetting of the feed with water followed by clean-up on Sep-Pak silica gel and OASIS HLB cartridges. Analysis of extracts was carried out on an Inertsil ODS-2 column with acetonitrile-water-formic acid as the mobile phase and UV detection at 230 nm. The limit of quantification (LOQ) of the method was 2.7 mg kg(-1). The proposed method was validated at a target species dependent minimum required performance limit (MRPL), at 2MRPL and at 5MRPL levels in pig, calf, piglet, sow, poultry, cattle and laying hen feeds. Recoveries at target species dependent MRPL levels ranged from 38 to 67%, within-day repeatabilities from 7 to 19% and within-laboratory reproducibilities from 13 to 27%. The proposed UV method is primarily suitable for screening purposes at subadditive levels, but semi-quantitative data can also be produced. Three MS detection modes (ion-source CID, full MS and MS2) were tested as an alternative and/or extension to UV detection. The selectivity and sensitivity of both LC-MS2 and LC-MS were much better than those of UV detection at 230 nm.

Polymyxin E-1 (colistin sulphate) (neuro-)intoxication in young ostriches ( Struthio camelus spp.).

Colistin (polymyxin E) is a cyclic polypeptide with a potent bactericidal action against most gramnegative bacilli. When used parenterally, polymyxins should be given with great care as they have a very small safety range, and easily induce neurotoxicity and nephrotoxicity. A dose of 39.5mg/kg body weight colistin sulphate injected subcutaneously induced rapid (within 1 to 3 h) mortality in young ostriches. Clinical signs of apathy, lethargy and hypotonia indicative of neurotoxicity of the compound were observed. At postmortem, vascular congestion of brain vessels was seen while, on histology, severe acute oedema was present in the epicardium and the intestinal serosa. Congestion of villi, swelling and vacuolization of the plexus ofAuerbach, as well as intermuscular and perivascular oedema of the heart, were also observed. In view of our observations in ostriches and in other species studied, a dose of >5mg/kg body weight polymyxin E is not considered safe for parenteral administration in ostriches.

Immunochemical detection of aminoglycosides in milk and kidney.

In 1996, the European Union established provisional maximum residue limits (MRL) for gentamicin, neomycin, streptomycin and dihydrostreptomycin in milk and tissue (0.1-5 mg kg-1). For the detection of these four aminoglycosides, three enzyme linked immunosorbent assays (ELISA) for applications in milk and kidney were developed. The screening of defatted and diluted milk resulted in limits of determination (LDM) of < 0.01 mg l-1. Kidney samples were deproteinized with a trichloroacetic acid solution (3%) and after filtration and the addition of buffer, aliquots were used in the ELISA. The LDM of the four aminoglycosides in kidney were < 0.05 mg kg-1. The ELISA were found suitable for the semi-quantitative screening of milk and kidney for the presence of the four aminoglycosides far below the MRL levels. In randomly taken milk samples (n = 776) and in kidneys derived from healthy pigs (n = 124), the aminoglycoside residues found were far below their established MRL. In eight out of the 94 kidney samples obtained from diseased animals after emergency slaughter, aminoglycoside residues were above the MRL.

Flubendazole residues in eggs after oral administration to laying hens: determination with reversed phase liquid chromatography.

Flubendazole residues in eggs were experimentally induced by providing groups of 8 laying hens feed with approximately 3, 10 and 30 mg kg-1 flubendazole for 21 days. Eggs were sampled during this period and one week after the administration. Samples of both whole egg and egg white/yolk were analysed separately. Flubendazole analysis was performed by reversed phase HPLC and UV detection at 250 nm (eggs) or 320 nm (feed). The limit of detection (LOD) for flubendazole in feed was 0.3 mg kg-1 and in whole egg 0.012 mg kg-1. Both the hydrolysed and reduced metabolites of flubendazole were also determined quantitatively. The eggs of control hens housed in the same room during the study period did not contain any detectable flubendazole or metabolite residue. The eggs from the lowest dosed group (3 mg kg-1 feed) did contain residues, but most of them were only slightly higher than the LOD. Residues in eggs collected from the laying hens that obtained feed with 10 and 30 mg kg-1 flubendazole reached a plateau level after some 10 days and there was a good dose response relation between levels in feed and those in eggs. The residues of parent compound and metabolites almost exclusively occurred in yolk, the metabolites accounting for some 60-65% of the total residue. The residues of the parent compound and its metabolites declined below 100 micrograms kg-1 5 days after the administration of dosed feed had ended.

Ivermectin detection in serum of onchocerciasis patients: relationship to adverse reactions.

Ivermectin treatment of onchocerciasis patients can be accompanied by adverse reactions. Not much is known concerning the pathogenesis of these reactions. Previous studies have demonstrated that the occurrence and extent of adverse reactions are related to infection intensity. However, some severely infected patients experience relatively few adverse effects. The aim of the present study was to investigate whether this seeming discrepancy could be due to diminished ivermectin absorption. Ivermectin concentrations one and two days after treatment were measured by high-performance liquid chromatography in sera of 71 skin snip-positive onchocerciasis patients (21 without reactions, 25 with mild reactions, 14 with moderate reactions, and 11 with severe reactions). The overall mean +/- SD ivermectin concentrations one and two days after a single oral dose (150 micrograms/kg) were 16.4 +/- 6.4 and 6.6 +/- 3.1 ng/ml, respectively. The overall mean +/- SD half-life was estimated to be 19.9 +/- 8.6 hr. The data presented did not show a relationship between ivermectin concentrations and the grade of adverse reactions.

Optimization and ruggedness testing of the determination of residues of carbadox and metabolites in products of animal origin. Stability studies in animal tissues.

A method developed for the determination of residues of carbadox and its metabolites in swine tissues using high-performance liquid chromatography with on-line precolumn enrichment and postcolumn derivatization with UV-VIS detection was optimized and the applicability of the method was extended to plasma and eggs. With the optimized method, more than twenty samples per person per day can be analysed. In the matrices investigated, the observed limit of determination for carbadox is 0.5-1 micrograms/kg and for desoxy-carbadox 0.5-2 micrograms/kg. The mean recovery for desoxy-carbadox in kidney, muscle and liver as established by two laboratories over a 2-month period is 95% (relative standard deviation = 14%, N = 37, 10 micrograms/kg). In other matrices the recoveries are between 83 and 91%. The recovery for carbadox is 70-80% in muscle, plasma and eggs. The method has been used routinely in pharmacokinetic and surveillance studies. Stability studies of kidney and liver samples spiked with carbadox showed that carbadox is rapidly decomposed (in vitro metabolism). After storage for about 1 h at 4 degrees C, more than 50% of the added amount is converted by reduction to desoxy-carbadox. In contrast, carbadox is stable in eggs and muscle under spiking conditions and during storage at -20 degrees C. Desoxy-carbadox is stable during spiking and storage at -20 degrees C in eggs and muscle. In kidney and liver, its stability was good under spiking conditions but could not be proved unequivocally during storage.

Liquid chromatographic multicomponent method for determination of residues of ipronidazole, ronidazole, and dimetridazole and some relevant metabolites in eggs, plasma, and feces and its use in depletion studies in laying hens.

A simple, rapid liquid chromatographic (LC) method that uses UV/VIS detection has been developed for the determination in eggs of residues of the histomonostats dimetridazole (DMZ), ronidazole (RON), ipronidazole (IPR), and side-chain hydroxylated metabolites of DMZ and RON. Sample pretreatment includes an aqueous extraction, purification with an Extrelut cartridge, and acid partitioning with isooctane. An aliquot of the final aqueous extract is injected into a reverse-phase LC system; detection is performed at 313 nm. The limits of determination are in the 5-10 microgram/kg range. A UV/VIS spectrum can be obtained at the 10 microgram/kg level by using diode-array UV/VIS detection. Recoveries are between 80 and 98% with a coefficient of variation of about 5%. Some 20 samples can be analyzed per day. A side-chain hydroxylated metabolite of IPR can also be detected with this method, as demonstrated with samples from animal experiments. After a single oral dose of the drugs to laying hens, residues of the parent compound and/or the hydroxylated metabolites could be detected in eggs 5-8 days after dosing. Plasma distribution and excretion in feces were established both with and without deconjugation. DMZ and IPR were extensively metabolized to hydroxylated nitroimidazole metabolites; RON was excreted mainly as the parent compound.

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.

Determination of residues of carazolol and a number of tranquilizers in swine kidney by high-performance liquid chromatography with ultraviolet and fluorescence detection.

A rapid and sensitive method has been set up for the determination of the beta-receptor blocker carazolol and the tranquillizers acepromazine, azaperone, chlorpromazine, haloperidol, propionylpromazine and xylazine in swine kidneys. The procedure involves extraction with acetonitrile, rapid sample clean-up with a Sep-Pak C18 cartridge and high-performance liquid chromatography with ultraviolet and fluorescence detection. The mean recoveries range from 93 to 101%, with the exception of xylazine (52%), and the coefficients of variation from 5.3 to 18.9% at a fortification level of 20 micrograms/kg. The limits of determination range from 0.3 micrograms/kg for carazolol to 1-10 micrograms/kg for the other drugs. The method has been tested in routine monitoring programmes.

Determination of residues of carbadox and some of its metabolites in swine tissues by high-performance liquid chromatography using on-line pre-column enrichment and post-column derivatization with UV-VIS detection.

A high-performance liquid chromatographic (HPLC) method that uses UV-VIS detection and post-column derivatization with sodium hydroxide was developed for the determination of the growth-promoting antibiotic carbadox and three of its metabolites in swine muscle, liver and kidney tissues. Sample pre-treatment involves extraction with methanol-acetonitrile, purification over an alumina-Florisil column and partition with isooctane. A 2-ml volume of the final aqueous extract is injected into a column-switching HPLC system; detection is performed at 420 nm. The limits of determination are in the range 1-5 micrograms/kg. Preliminary experiments show a good precision with mean recoveries of 81-87% and a coefficient of variation of 4-10%. The method is highly selective and can be used in routine monitoring programmes.

High-performance liquid chromatographic screening and confirmation methods for chloramphenicol residues in meat with off-line cartridge sample clean-up and on-line diode array UV-VIS detection.

Two high-performance liquid chromatography (HPLC) methods for the analysis and confirmation of residues of the antibiotic chloramphenicol in edible animal tissues are described. The first method consists of an aqueous extraction followed by purification through an Extrelut cartridge and toluene partition. With this simple and rapid method, meat samples can be screened at the 5 micrograms/kg level. The second, more comprehensive, method is based on ethyl acetate extraction, followed by purification through a silica Sep-Pak cartridge and partition with buffer-diethyl ether and water-toluene. Confirmation of positive peaks at the 10 micrograms/kg level is performed by diode array UV-VIS detection. The recoveries for the two methods at the 10 micrograms/kg level are 58 and 85% respectively, coefficients of variation 5-6%. With the confirmation method, glucuronide and sulphate conjugates can be determined. However, in a positive reference sample (pig) none was observed.

Procedure for the gas chromatographic-mass spectrometric confirmation of some exogenous growth-promoting compounds in the urine of cattle.

As gas chromatography-mass spectrometry is the most conclusive confirmation technique available today for the detection of ppb levels of anabolics in the urine of cattle, the following procedure was used. The urine is hydrolysed with Helix pomatia intestinal juice, extracted, and the extract cleaned by gel-permeation chromatography or with Extrelut. In one fraction are eluted diethylstilboestrol, dienoestrol, hexoestrol, methyltestosterone, ethinyloestradiol, zeranol and trenbolone. This fraction is injected into a two-dimensional high-performance liquid chromatography system. From the first column, the fraction containing the above-mentioned compounds is transferred to the second column, where a separation into two fractions is obtained. The first fraction contains zeranol and trenbolone, the second fraction the stilbenes, methyltestosterone and ethinyloestradiol. In general, the compounds are derivatized with an N,O-bis (trimethylsilyl)trifluoroacetamide-trimethylchlorosilane mixture to a trimethylsilyl derivative. With stilbene, confirmatory derivatization into heptafluorobutyryl derivatives is necessary. In combination with a Finnigan 4000/INCOS system, a CP-Sil-5 CB and a CP-Sil-19 CB capillary are used for final confirmation. Two capillaries with different polarities are necessary to overcome problems with possible interferences from compounds extracted from the urine. Recoveries at the ppb level are better than 80%.

Quantitative determination of specified chlorobiphenyls in fish with capillary gas chromatography and its use for monitoring and tolerance purposes.

A gel permeation (GPC) clean-up procedure of fish samples in combination with capillary gas chromatography is used for the determination of individual chlorobiphenyls. The described method is compared with a more universal method based on saponification. Starting from a tolerance for PCB's expressed as a technical Aroclor, tolerance for some specified individual chlorobiphenyls are derived. It is shown that these tolerances for specified individual chlorobiphenyls can be used for monitoring and tolerance purposes. Rejection obtained with the Dutch PCB tolerance agree with rejections obtained with individual tolerances.

Quantitative determination of individual chlorinated biphenyls in milkfat by splitless glass capillary gas chromatography.

A method is described for determining individual polychlorinated biphenyls (PCBs) in milkfat at the ppb level. Extraction and major cleanup are done simultaneously by saponification of the milkfat. After final cleanup on basic alumina, the concentrated extract is injected under splitless conditions on a capillary column coated with CPSil 7 stationary phase. The recovery of several individual PCBs from milkfat at a level of 25 ppb is better than 80%. The reproducibility for duplicates analyzed on several days showed standard deviations from 0.2 to 0.9 ppb for 19 individual chlorinated biphenyls. Some preliminary results for 165 samples of milkfat are reported.