Analytical strategies for the screening of veterinary drugs and their residues in edible products.

The development of analytical strategies for the regulatory control of drug residues in food-producing animals is discussed. Analytical methods for the determination of veterinary drugs in edible products are based on microbiological, immunochemical and physicochemical principles. Because of complexity of biological matrices such as egg, milk and meat, well designed, and often sophisticated, off-line or on-line sample treatment procedures are essential, especially when utilising physicochemical multi-residue screening procedures. Since large series of samples have often to be analysed, automation is increasingly becoming important. Confirmation of the identity of drug residues and validation of the analytical results implies the use of adequate analytical methods. In its turn, this requires well established criteria for those methods and/or equivalent reference methods.

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.

On-line combination of dialysis and column-switching liquid chromatography as a fully automated sample preparation technique for biological samples. Determination of nitrofuran residues in edible products.

The potential of dialysis coupled on-line with trace enrichment by column-switching high-performance liquid chromatography as an automated sample treatment technique in drug residue analysis has been investigated. The nitrofuran veterinary drugs furazolidone, nitrofurazone, nitrofurantoin and furaltadone were used as model compounds. Critical parameters, i.e., dialyser dimensions, air segmentation, dialysis time, flow-rates and enrichment column breakthrough, were evaluated. Depending on the analytical purpose, the technique can be set up in either a highly sensitive or a high-speed mode. High dialysis efficiencies (greater than 85%) can be obtained in a stopped-flow dialysis of only 3 min. Aqueous biological sample volumes (eggs, meat, milk) of 100 microliters to 4 ml can be injected with only sample treatment. A routine monitoring method for residues of nitrofuran drugs in edible products was set up. A 4-ml sample was dialysed in a pulsed mode with an efficiency of about 30% and concentrated on a short C18 column. Recoveries compared with standards were 75-85% (coefficient of variation 2-7%). Limits of determination ranged from 1 to 10 micrograms/kg. At this concentration level, ca. 30 samples can be monitored per day.

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 sulfadimethoxine, sulfamethoxazole, trimethoprim and their main metabolites in porcine plasma by column switching HPLC.

A HPLC method for the determination of sulfadimethoxine, sulfamethoxazole, trimethoprim and their main metabolites in porcine plasma is reported. The metabolites under investigation were the N4-acetyl sulfonamides and 3'- and 4'-demethyl trimethoprim. In order to obtain a sensitivity of 25-50 ng ml-1, the application of column switching HPLC was investigated. An on-line preconcentration of the drugs and metabolites was preceded by an off-line sample pre-treatment. Parent compounds and metabolites were separated by reversed-phase HPLC followed by UV-detection. The mean recoveries for 4'-demethyl trimethoprim were greater than 80% while the mean recoveries for the other compounds were greater than 90%. Application of the method for analysis of plasma samples obtained from pharmacokinetic studies is described.

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.

Monitoring milk for chloramphenicol residues by an immunoassay (Quik-card).

An easy to perform immunoassay method (Quik-card) for monitoring milk on the presence of chloramphenicol residues exceeding 5 to 10 ppb is described, as well as an extraction and preconcentration method utilising a Sep-pak silica cartridge, which enables the detection of CAP residues in milk in concentrations exceeding 1 ppb by HPLC and 0.1 ppb by the immunoassay.

The effect of dietary nitrite and nitrate on the metabolism of sulphadimidine administered orally to pigs.

The in vivo interaction of sulphadimidine (SDM) with nitrite and nitrate has been investigated in pigs. It was shown that the combined oral treatment with SDM and nitrite but not nitrate leads to the formation of a deaminated compound, which becomes the major metabolite in plasma soon after cessation of the treatment. The major in vitro reaction product, 1,3-di(4-[N(4,6-dimethyl-2-pyrimidinyl)]-sulphamoylphenyl)-triazen e, DDPSPT as has been reported previously, could not be detected in blood, urine or faeces of the exposed animals. No effect of nitrite or nitrate could be observed on the acetylation of SDM.

Plasma disposition and renal clearance of sulphadimidine and its metabolites in laying hens.

Plasma disposition of sulphadimidine (SDM) and its metabolites was studied in laying hens after 100 mg SDM kg-1 doses were administered as a single intravenous dose, a single oral dose and multiple oral doses once daily for five consecutive days. SDM was extensively metabolised by acetylation and hydroxylation. In plasma, the metabolite observed with the highest concentration was N4-acetylsulphadimidine (N4-SDM) followed by hydroxymethylsulphadimidine (CH2OH) and 5-hydroxysulphadimidine. Following intravenous administration a biphasic elimination (as seen for a capacity limited reaction) pattern for SDM and its metabolites was observed. Multiple (5x) SDM dosing revealed plasma SDM concentrations ranging between 7 and 108 micrograms ml-1; within 96 hours of termination of the multiple SDM dosing, the plasma SDM concentration was below 0.01 micrograms ml-1. The renal clearances of N4-SDM and the hydroxy metabolites were approximately 10 times greater than that of SDM. The SDM mass balance (faecal/urinary recovery) showed a loss of 56 per cent after intravenous dosage and of 67 per cent after a single oral dosage; the hydroxy metabolites accounted for the highest percentage in faeces/urine. Thus additional metabolic pathways must exist in laying hens.

Monitoring of veterinary drug residues by a combination of continuous flow techniques and column-switching high-performance liquid chromatography. I. Sulphonamides in egg, meat and milk using post-column derivatization with dimethylaminobenzaldehyde.

By using a combination of a low-pressure continuous flow module and a column-switching high-performance liquid chromatographic system, procedures have been developed for the automated residue analysis of sulphonamides and selected other drugs in meat, egg and milk. Aqueous extracts are purified by on-line dialysis and subsequent trace enrichment on a short column containing silica-based or polymer material. After backflush to the analytical column, detection is performed either at 280 nm or, after post-column derivatization with p-dimethylaminobenzaldehyde, at 450 nm. With these procedures the fully automated determination of both polar and apolar sulphonamides and dapsone above 5-20 micrograms/kg is possible. Coefficients of variation are 4-10%, recoveries compared to standards are 85-90%. The methods developed were tested in routine veterinary drug control.

Some pharmacokinetic data about furaltadone and nitrofurazone administered orally to preruminant calves.

A single oral dosage of furaltadone and nitrofurazone (14.0 mg/kg) to 5 preruminant calves (in a cross-over trial) revealed mean maximum plasma concentration of 2.5 and 3.5 microgram/ml, respectively, at approximately 3 h after administration. The final elimination half-lives of furaltadone and nitrofurazone were 2.5 and 5 h, respectively. Urinary recovery of these two nitrofurans in 3 calves revealed approximately 2% of the orally administered dose. The renal clearance of the unbound drugs did not differ (for both drugs approximately 0.42 ml/min/kg); furaltadone clearance was strongly related to urine flow.

Rapid sample preparation methods for analysis of residues of sulfamethazine and its N4-acetyl and desamino metabolites in swine tissue by HPLC.

Rapid sample preparation methods for the determination of sulfamethazine (SMZ) and its N4-acetyl and desamino metabolites in swine tissues at the 0.1 mg kg-1 level are presented. The methods use sonication-aided extraction with dichloromethane. For SMZ and N4-acetyl SMZ analysis extracts are cleaned up and concentrated on a silica disposable column followed by HPLC on a CP Spher C8 column using acetonitrile-sodium acetate buffer as the mobile phase. For desamino-SMZ analysis the extract was cleaned up and concentrated on a Florisil disposable column, followed by HPLC on a Nucleosil 5-CN column after formation of an ion-pair complex with 1-heptanesulfonic acid. For desamino SMZ peak identification by diode-array UV/VIS detection is also described. Mean recoveries from spiked tissue samples were about 87% (muscle) and 76% (kidney) for SMZ and N4-acetyl SMZ and about 70% for desamino SMZ.

Residues of sulphadimidine and its metabolites in eggs following oral sulphadimidine medication of hens.

The depletion of sulphadimidine (SDM) and its N4-acetyl and hydroxy metabolites was studied in eggs laid by hens after administration of either a single or multiple oral dosages of 100 mg SDM/kg. During medication and until 1 day after the last dose, the SDM and its metabolite concentrations in the egg white exceeded those in the egg yolk and reflected the plasma levels. In the period starting 2 days after the (last) dosage, the SDM concentration in the yolk became higher than in the egg white, and the drug depletion curves ran parallel. The mean maximum amount of SDM found in the whole egg was 1500 micrograms after a single and 1280 micrograms after multiple dosage. In eggs, traces of the N4-acetyl and 6-methylhydroxy metabolites could be detected (mainly in the egg white), and their concentrations were approximately 40 times lower than those of the parent drug. A highly significant correlation (P less than 0.005) was found between the development stage of the oocyte at the time of (last) medication and the amount of SDM found in the egg that developed from it. A period of 7 or 8 days after the (last) dosage of 100 mg SDM/kg/day is required to obtain SDM levels below 0.1 micrograms/g egg.

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.

Determination of furazolidone residues in eggs by HPLC followed by confirmation with a diode-array UV/Vis detector.

A sensitive HPLC method for the determination of furazolidone residues in eggs (10-1,000 micrograms/kg) is described. Recovery is about 86%. With the aid of a UV/Vis Diode-Array detector confirmation up to the 15-ppb level was possible. In order to test this method with "real" samples, three laying hens received 30 mg each of furazolidone in feed (single dose). The eggs were collected for five days. After five days traces of furazolidone (5 micrograms/kg) could still be detected.