Development and validation of an improved method for confirmation of the carbadox metabolite, quinoxaline-2-carboxylic acid, in porcine liver using LC-electrospray MS-MS according to revised EU criteria for veterinary drug residue analysis.

A method is described for the quantitative determination of quinoxaline-2-carboxylic acid (QCA), the marker residue for the veterinary drug carbadox, in swine liver. Tissue is subjected to alkaline hydrolysis followed by liquid-liquid extraction. QCA residues are cleaned up using automated solid phase extraction (SPE), before a final liquid-liquid extraction step. Analysis is based on LC coupled to positive ion electrospray MS-MS, monitoring product ions at m/z 129, 102 and 75 for QCA and at m/z 106 for the internal standard (d4-QCA). The method has been validated according to draft revised EU criteria for analysis of veterinary drug residues, and is suitable for monitoring tissues taken under national surveillance schemes. The method has been validated at 3, 10, 30, 100 and 300 microg kg(-1). The method performance characteristics, CCalpha (decision limit) and CCbeta (detection limit) were determined to be 0.16 and 0.27 microg kg(-1), respectively. The described method, which is relatively rapid and applicable to large sample numbers, correlates well (r2 = 0.9799) with a widely used GC-MS assay for QCA.

Effect of various lithotomy positions on lower-extremity blood pressure.

BACKGROUND: Compartment syndrome of a lower extremity from hypoperfusion is a rare but potentially devastating complication of the lithotomy position during surgery. The aim of this study is to determine the effects of various lithotomy positions on lower-extremity blood pressures. METHODS: Blood pressure in eight young, healthy people was studied for 10 lithotomy positions. Blood pressure measurements were taken in both the upper arm (brachial artery) and the lower extremity (dorsalis pedis). The heart-to-ankle height gradient in each position was measured, and a predicted lower-extremity systolic pressure was calculated. The measured and predicted lower-extremity systolic blood pressures were compared with repeated measures analysis of variance. RESULTS: As a group, the mean systolic blood pressures in the lower extremities correlated closely with the predicted values. However, the difference between measured and predicted pressures varied among the 10 positions (P < 0.05). CONCLUSIONS: Although lower-extremity systolic blood pressures in the young, healthy volunteers correlated with predicted values, there was an additional reduction in pressure associated with the lithotomy position. This surprising finding suggests that a lengthy procedure necessitating the use of a lithotomy position for only a portion should be planned so the remainder of the procedure can take place before establishing the position or so the position can be changed to an alternative position when it is no longer needed.

Biliary elimination of endogenous nortestosterone by pregnant cows.

A temporal study of the biliary elimination of endogenous 19-nortestosterone during two successive pregnancies was made in three cows with cannulated gall bladders. Bile samples were analysed for 17 beta-19-nortestosterone (beta-NT) and the 17 alpha-epimer (alpha-NT) by using high resolution gas chromatography and mass spectroscopy. No beta-NT was detected in any of the samples analysed. However, alpha-NT was detected from around 120 days of gestation in each of the cows. Peak concentrations were observed in the last week before calving and ranged from 9.5 to 36.7 ng/ml. After parturtion, the concentrations of alpha-NT declined rapidly and were undetectable by seven days after calving, and it was not detected again until after 120 days of gestation. The biliary concentrations of alpha-NT detected subsequently were similar to those observed in cattle several weeks after an exogenous injection of the synthetic ester beta-NT phenylpropionate.

Zeranol is formed from Fusarium spp. toxins in cattle in vivo.

Zeranol, a semi-synthetic oestrogenic growth promoter, was banned in the EU in 1988. The ability of Member States to police the ban on zeranol has been hampered by suggestions from New Zealand and from this laboratory that zeranol may be formed by the in vivo metabolism of naturally occurring Fusarium spp. toxins. The present study demonstrates that zeranol is formed from alpha-zearalenol and zearalenone in vivo and is detected in bovine bile following the oral administration of these compounds. However, it is not detected following administration of beta-zearalenol. These data suggest that hydrogenation of alpha-zearalenol, probably in the rumen, is responsible for the appearance of zeranol. The present study shows that environmental contamination with Fusarium spp. toxins is widespread in Northern Ireland. Fusarium spp. toxins were present in 32% (n = 422) of all bovine bile samples tested for zeranol during 1995. Zeranol itself was confirmed in 6.6% (n = 28) of the samples. However, the mean alpha-zearalenol and beta-zearalenol concentrations in the bile of zeranol-positive animals were 12 and 9 times higher, respectively, than those in the zeranol-negative animals. The alpha-zearalenol concentration always exceeded the zeranol concentration by at least 5:1. This may, in the future, permit differentiation between zeranol abuse and natural contamination.

Use of liquid chromatography-mass spectrometry in the analysis of residues of antibiotics in meat and milk.

The advent of affordable LC-MS systems has led to a massive increase in a number of publications describing quantitative methods for the analysis and confirmation of veterinary drug residues. The lack of volatility and thermal instability of many antibiotics makes LC-MS the method of choice for their analysis. In the review, analytical methods for the determination of residues of each of the major classes of antibiotics are presented.

Iso- and epi-iso-chlortetracycline and the principal metabolites of chlortetracycline in the hen's egg.

Chlortetracycline (CTC) is a broad spectrum antibiotic, licensed for use without any withdrawal period, in chickens laying eggs intended for human consumption. In the European Union, a maximum residue limit (MRL) in eggs of 200 microgram/kg for the sum of the concentrations of CTC and its 4-epimer (4-epi-CTC) has been established. Two major CTC metabolites have been identified in eggs. These compounds, iso-CTC and 4-epi-iso-CTC, have never previously been shown to be significant products of CTC metabolism in poultry or in any other species. The total amount of CTC present in eggs, as all of the chemical forms measured, can exceed the MRL by anything up to a factor of four (170-820 micrograms/kg).

Metabolism of chlortetracycline: drug accumulation and excretion in the hen's egg.

Chlortetracycline (CTC) is one of the few antibiotics that can be used without any withdrawal period in chickens laying eggs intended for human consumption. 6-Iso-CTC and 4-epi-6-iso-CTC have recently been identified as the principal metabolites of CTC in eggs. Although not covered by the European Union (EU) maximum residue limit (MRL) for CTC, these compounds, taken together, accumulate in the eggs of birds treated therapeutically with CTC to a mean concentration equivalent to more than twice the EU MRL (200 micrograms kg-1) in eggs. Plateau concentrations in eggs were achieved after approximately 3 d of medication. Following withdrawal of medication, mean egg concentrations of these compounds were maintained for 48 h, before falling below a level equivalent to the MRL after 5 d. Feeds containing typical sub-therapeutic contamination concentrations of CTC did not produce mean concentrations of 6-iso-CTC plus 4-epi-6-iso-CTC, combined, greater than 200 micrograms kg-1. It is not known whether these compounds are formed as a result of metabolism or of chemical degradation. However, analysis of ovules pre-lay showed that all of the CTC present in this matrix was in the form of 6-iso-CTC and 4-epi-6-iso-CTC, and not as the parent drug. Although microbiologically inactive, the toxicological properties of 6-iso-CTC and 4-epi-6-iso-CTC are not known.

Use of biosensors for rapid drug residue analysis without sample deconjugation or clean-up: a possible way forward.

The drug salbutamol (SBL) is a beta-agonist that may be used illegally as an animal growth promoter. SBL is also a good example of a drug which is excreted in the form of glucuronides and sulfates. Such metabolites cause complexities in analysing for the presence of drug residues. In the majority of cases a process of deconjugation and sample clean-up is required prior to analysis. This is both time consuming and causes some loss of accuracy. In this study, the urine of calves treated with SBL orally for 3 d was collected during and after medication. Samples were assayed before and after hydrolysis by two different methods, radioimmunoassay (RIA) and a newly developed biosensor immunoassay (BIA). Some samples were also analysed by GC-MS. The results clearly showed that both screening assays (RIA and BIA) found high concentrations of SBL residues throughout the study. This was especially true in the BIA method. It was also demonstrated that urine sample analysis without the need for deconjugation or clean-up could be achieved. Results obtained by GC-MS tended to be an order of magnitude lower than the corresponding screening test results. This work showed that biosensor based veterinary drug residue testing procedures can be developed which can generate results in real time without the need for time consuming sample preparation.

Monensin carry-over into unmedicated broiler feeds.

European Union Member States are now required to monitor poultry meat for the presence of coccidiostat residues. Among other factors contributing to the production of residue-free food is the ability of animal feed manufacturers to produce medication-free feedstuffs, ensuring the proper observance of withdrawal periods prior to slaughter. Carry-over of medication was investigated in a local poultry feed mill that was using monensin as its principal coccidiostat for broilers. Monensin, at levels in excess of 5% of the therapeutic dose (approximately 110 mg kg-1), was present in 22.5% of 40 withdrawal feeds. Subsequent studies in the mill indicated that most of the contamination occurred during the processing of feeds after the mixing stage. The mill altered its manufacturing process as a result of this study. The consequence of this was that the incidence of monensin withdrawal feeds, at levels greater than 5% of the therapeutic dose, fell from 22.5 to 2.5%. This collaborative study has helped the feed compounder to produce more effective withdrawal feeds, thereby reducing the potential exposure of consumers to unwanted residues of monensin in poultry meat.

Comparison of conventional immunoassays and the oestrogen radioreceptor assay for screening for the presence of oestrogenic anabolic compounds in urine samples.

Screening for the presence of anabolic growth promoters in urine samples from cattle grown for meat production can be performed by (semi)quantitative methods such as immuno-, receptor- or cell-based assays or by quantitative methods with mass spectrometric detection which can also include confirmation of compounds. In this study conventional immunoassays used at two different institutes [Veterinary Sciences Division (VSD) in Northern Ireland and TNO Nutrition and Food Research Institute (TNO) in The Netherlands] were compared with the oestrogen radioreceptor assay (ORRA), with GC-MS as the reference method. Urine samples were generated by treating calves (n = 2 per group) intramuscularly with ethynyloestradiol (EE2), diethylstilbestrol (DES) or alpha-zearalanol (zeranol, ZER). Urine samples were collected up to 21 d after administration of the oestrogenic compounds. Samples were screened by enzyme immunoassay or radioimmunoassay and by the ORRA and also by GC-MS. Values found by VSD were lower by a factor of 1-20 than those measured by TNO. These differences could be explained by differences in sample clean-up (immunoaffinity chromatography versus solid-phase extraction) and by differences in cross-reactivities between the antisera used. The ORRA and GC-MS showed similar results for EE2 and DES, but produced lower results (by a factor of ca. 3) for ZER owing to the relatively low affinity of ZER for the oestrogen receptor. The most important finding was that the withdrawal period for calves treated with EE2, DES or ZER was similar for each of the screening methods used. Therefore, it is concluded that the choice of screening method does not affect the probability of finding a positive sample.

Screening for chloramphenicol residues in the tissues and fluids of treated cattle by the four plate test, Charm II radioimmunoassay and Ridascreen CAP-Glucuronid enzyme immunoassay.

The administration of chloramphenicol (CAP) is banned in food animals in the European Union (EU). It is, therefore, important to have adequate screening methods to determine if residues of CAP and its major metabolite, chloramphenicol-glucuronide (CAP-Gluc), are present in samples taken for monitoring purposes. Six castrated male cattle were treated with a single intramuscular injection of 10 mg kg-1 CAP. Animals were sampled once daily for urine and were slaughtered at 3 and 6 d post-injection. Samples of bile, kidney, liver and diaphragmatic muscle were removed at slaughter. All matrices were analysed using the four plate test (FPT) bioassay, the Charm II radioimmunoassay and a Ridascreen CAP-Glucuronid competitive enzyme immunoassay (EIA). The FPT detected CAP residues in urine samples taken up to 2 d post-treatment. The Charm assay detected CAP in the urine for up to 4 d post-treatment. The EIA detected CAP throughout the 6 d sampling period. Samples of bile were positive by both the EIA and the Charm assay at day 3 and day 6. No zones of inhibition were obtained using the FPT in bile or diaphragm either with or without sample pre-treatment with beta-glucuronidase. However, the kidney and the liver from one animal killed at day 6 gave larger zones of inhibition after treatment with beta-glucuronidase, indicating the presence of CAP. The kidneys of all treated animals slaughtered at day 3 were positive by both the EIA and the Charm assay but none of the kidneys at day 6 tested positive by either method. Owing to technical difficulties, the Charm assay was not suitable for the analysis of liver. The EIA failed to detect CAP in the liver of any treated animal. It is concluded that urine appears to be the best matrix for screening purposes. The sensitivity of the FPT is inadequate for the determination of CAP residues were minimal withdrawal periods have been observed. The Charm assay and the EIA were suitable for the detection of both CAP and CAP-Gluc in tissues and body fluids for longer periods post-administration. The EIA was more sensitive for the determination of low concentrations of CAP and its metabolite.

Determination of trimethoprim in tissues using liquid chromatography-thermospray mass spectrometry.

A method is described for the determination of the antibacterial drug trimethoprim in tissues. Minced tissue is homogenised with chloroform-acetone (1 + 1 v/v), filtered, and the filtrate evaporated to an oily residue using a rotary evaporator. The residue is redissolved in methanol-water-acetic acid (50 + 48.7 + 1.3 v/v) and any fats present are partitioned into hexane. The aqueous phase is analysed by liquid chromatography-thermospray mass spectrometry in positive mode with the protonated molecular ion at m/z 291 being monitored. Recoveries ranged between 60% in liver and 79% in muscle. The limit of determination was 25 micrograms kg-1 and the limit of detection was approximately 4 micrograms kg-1. The method is suitable for monitoring tissues taken under national surveillance schemes for veterinary drug residues.

Gas chromatographic-mass spectrometric determination of sulfamethazine in animal tissues using a methyl/trimethylsilyl derivative.

A method is described for the determination of sulfamethazine in swine tissues by GC-MS. Samples are extracted with chloroform-acetone, followed sequentially by two solid-phase clean-up steps using silica gel and SCX ion exchange. The extracts are then partitioned between sodium dihydrogenphosphate (0.1 mol l-1) and methyl tert-butyl ether, the organic phase is evaporated to dryness and the sulfamethazine subjected to a double derivatization via methylation and silylation and determined by GC-MS in the selected-ion monitoring mode. Quantification is achieved by measuring the ratio of the abundances of the M-65 (-HSO2) ions of the derivatives of sulfamethazine and the internal standard, [phenyl-13C6]sulfamethazine, at m/z 299 and 305, respectively. The presence of sulfamethazine can be confirmed using the abundance ratios of the ions of m/z 299, 300 (-SO2) and 349 (-CH3). Recovery values from muscle, kidney and liver spiked at 0.05, 0.2 and 0.4 ppm ranged from 86 to 114% with RSDs between 2.8 and 9.0%. The limit of detection for the assay is 0.01-0.02 ppm. The methyl/trimethylsilyl derivatives exhibited better chromatography than the commonly used N1-methyl derivatives; for the same conditions, the peak was sharper and tailing was significantly reduced.

The production of pig tissue sulphadimidine reference material.

The production of stable homogeneous reference materials containing the antimicrobial agent sulphadimidine in pig tissue is described. These were commissioned by the Community Bureau of Reference (BCR), established by the Commission of the European Communities, to promote improvements in analytical accuracy and to ensure uniformity of results determined by member states. Sulphadimidine-containing tissue powders (400 vials each of muscle, liver and kidney) were prepared by orally dosing pigs with drug, producing lyophilized tissue powders and blending these with negative tissues from unmedicated animals to achieve target concentrations. Details of the production process, the stabilizing procedure developed and the analytical assessments of homogeneity and stability are given.

Possible naturally occurring zeranol in bovine bile in Northern Ireland.

Zeranol and two Fusarium toxins, alpha-zearalenol and beta-zearalenol, were confirmed by gaschromatography/mass spectrometry (GC/MS) in bovine bile samples referred to this laboratory for analysis. No evidence of zeranol abuse was found on-farm. Given the recent suggestion that zeranol might arise from the metabolism of these Fusarium toxins, and the finding of zeranol in bovine and ovine urine across the EU, it was concluded that the residues had arisen as a result of natural metabolism.

Sensitive gas chromatographic-high resolution mass spectrometric method for the determination of methylmalonic acid in bovine plasma.

A sensitive and highly specific method for the determination of methylmalonic acid (MMA) in bovine plasma is described. Following solvent extraction and butylation, samples are analysed by gas chromatography and detected using high-resolution mass spectrometry. The limit of detection for the assay was 0.025 mumol l-1 MMA and the recovery of added MMA ranged from 98 to 103%. The application of the method is demonstrated for the analysis of MMA in plasma taken from cattle that had been maintained on a cobalt-deficient diet for 64 weeks.

Comparison of the determination of four sulphonamides and their N4-acetyl metabolites in swine muscle tissue using liquid chromatography with ultraviolet and mass spectral detection.

A discharge-assisted LC-MS method has been developed and validated for the analysis of four sulphonamides (sulphathiazole, sulphadiazine, sulphamerazine and sulphadimidine) and their N4-acetyl metabolites in the muscle of swine treated with Polysulpha-Complex, which contains all four drugs. The clean-up procedure developed involved chloroform-acetone extraction followed by Sep-Pak silica solid-phase extraction. In parallel a LC-UV method was validated using the same clean-up procedure. Blank tissue was fortified at levels between 20 and 100 micrograms/kg. [13C]sulphadimidine was used as internal standard. The samples were analysed with thermospray LC-MS. The [M + H]+ ion was the major ion in all cases and was employed for single-ion monitoring. The limits of detection (LOD) were below 25 micrograms/kg and the limits of quantification (LOQ) for most sulphonamides were ca. 100 micrograms/kg. Incurred muscle tissues were measured by both LC methods and the concentrations of the sulphonamides were found to be similar. However, the LC-MS procedure is more suitable for confirmatory analysis due to its specificity.

Efficacy, pharmacokinetics and effects on egg-laying and hatchability of two dose rates of in-feed fenbendazole for the treatment of Capillaria species infections in chickens.

Laying broiler breeder hens infected with Capillaria obsignata were treated with in-feed fenbendazole either at a dose of 30 ppm for six days or 80 ppm for three days. The respective efficacies of treatment were 92.3 and 99.3 per cent. These results were a reflection of the plasma pharmacokinetics of fenbendazole and its metabolites in the regimens, which showed that the detectable plasma concentrations of the anthelmintically active fenbendazole sulphoxide in the second treatment were significantly greater than the first, with the area under the curve 1.34 times that of treatment 1. Both treatments produced a slight decline in egg production, but only the loss in production with treatment 1 was significant. Hatchability of eggs with both treatments was not significantly affected.