Determination of morantel-related residues in bovine milk by electron capture gas chromatography

A gas chromatographic assay was developed to determine major residues of morantel in bovine milk over a range that is suitable for monitoring residues of the drug. The method is based on hydrolysis of the N-methyl-tetrahydropyrimidine portion of morantel and its metabolites to N-methyl-1,3-propanediamine, and converting the diamine to an N,N-bis-(2-nitro-4-trifluoromethylphenyl) derivative. The addition of an internal standard, the N-desmethyl-N-ethyl homolog of pyrantel, to the milk sample circumvents any potential problem that could arise from variable reaction yields, and eliminates the true recovery as a factor affecting the accuracy and precision of the procedure. The concentrations of the derivatives are determined by pulsed electron capture gas chromatography over a linear dynamic range that is equivalent to 12.5-50 ppb morantel. The method was evaluated at the 0, 12.5, 25, and 50 ppb levels in fortified bovine milk, and in a withdrawal sample containing physiologically incurred morantel residues. Mean values of 14 +/- 1.7, 24 +/- 3.7, and 47 +/- 6.9 were found for the fortified samples, approximately 3 ppb for control milk, and 16 +/- 1.7 ppb for the withdrawal sample.     

Liquid chromatographic determination of multiple sulfonamide residues in bovine milk

A liquid chromatographic method has been developed for simultaneous determination of residues of 10 sulfonamide drugs at 10 ppb and above in raw bovine milk. The method is based on a chloroform-acetone extraction, evaporation of organic phase, dissolution of residues in an aqueous potassium phosphate solution, and extraction of fatty residue into hexane. The aqueous layer is collected, filtered, injected onto an LC system, and detected by ultraviolet absorption at 265 nm. To elute all 10 sulfonamides isocratically, 2 chromatographic conditions are required. Seven sulfonamides can be quantitated with 12% methanol in the mobile phase; 4 sulfonamides can be quantitated with 30% methanol. Sulfamethazine, the most widely used sulfonamide, is detected on both systems. Recoveries are 44-87% for individual sulfonamides, with only 2 below 60%. Coefficients of variation are 3-13% at 10 ppb.     

Neutral cleanup procedure for 2,3,7,8-tetrachlorodibenzo-p-dioxin residues in bovine fat and milk.

A neutral cleanup method for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in milk and animal tissue was developed involving solvent extraction and liquid adsorption chromatography on magnesia-Celite 545, alumina, and Florisil. Cleaned up extracts were subjected to dual-ion analysis in a direct probe high resolution mass spectrometer, interfaced to a multi-channel analyzer for signal averaging. Calibration experiments were carried out with bovine milk and beef fat samples containing added TCDD. The 37CI isotopic isomer of TCDD was added as an internal standard. The response was linear for concentrations in the ppt range, with recoveries about 80%. Milk from a cow fed TCDD was cleaned up by the neutral procedure or, alternatively, a base-acid extraction procedure. The TCDD recoveries for both procedures were essentially the same. Recoveries of TCDD from liver samples of a rat given 14C-TCDD intraperitoneally, subjected to neutral cleanup and radioactive counting, were about 70%.     

Development of an indirect competitive ELISA for flumequine residues in raw milk using chicken egg yolk antibodies

To detect flumequine in raw milk, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed. By carbodiimide conjugation, flumequine was conjugated to cationized bovine serum albumin (cBSA-flumequine) and to cationized ovalbumin (cOVA-flumequine). For the immunization of chickens, cBSA-flumequine was used, which allowed the isolation of specific chicken egg yolk immunoglobulins (IgY) for flumequine. As the coating antigen in the immunoassay, cOVA-flumequine was used. In the indirect competitive assay, standard flumequine was incubated together with the anti-flumequine antibodies. The antibody by which the lowest concentration of free flumequine that gives 50% inhibition of binding (IC50) was found in aqueous dilution was further tested for the applicability to detect flumequine in raw milk. An IC50 level in milk was reached that was about 5 times lower than in aqueous solution. So flumequine can be detected directly in raw milk at maximum residue level (50 microg/kg). No cross-reactivity was noticed with various related quinolones.     

Determination of thiabendazole, 5-hydroxythiabendazole, fenbendazole, and oxfendazole in milk

The liquid chromatographic determination previously developed for benzimidazoles in cattle liver has been slightly modified and applied to the determination of 4 benzimidazoles in milk. Recoveries of fenbendazole (FBZ), oxfendazole (OFZ), and thiabendazole (TBZ) from milk fortified at the 10 ppb level were 80% or greater with an intralaboratory coefficient of variation of 11% or less. Recovery of 5-hydroxythiabendazole (5-OH-TBZ) at the 30 ppb level averaged 56% with an intralaboratory coefficient of variation of 5%. Limited data on the depletion of FBZ, OFZ, TBZ, and 5-OH-TBZ in milk were also generated.     

固相萃取-高效液相色谱-质谱联用检测牛奶中地塞米松残留

建立了固相萃取-高效液相色谱-质谱联用检测牛奶中地塞米松残留的方法.牛奶样品经乙酸乙酯提取,固相萃取柱净化,浓缩过滤后上机测定,标准加入法定量.结果显示,地塞米松在1～50ng/mL范围内,线性关系良好,相关系数r大于0.999,3个水平的平均加标回收率为65.7％～85.7％,相对标准偏差小于10％,方法定量限和检出...     

酶联免疫法快速筛查牛奶中的二噁英

二噁英是最受人们关注的持久性有机污染物之一。本研究建立了ELISA快速筛查牛奶基质中二噁英的检测方法。牛奶样品经真空冷冻干燥,加速溶剂萃取法萃取、用酸性硅胶柱、活性炭柱净化后,浓缩氮吹后复溶,进行ELISA检测。结果表明,方法回收率为72.0%~125.2%,相对偏差<30%。本法对牛奶中二噁英毒性当量(TEQ)测定值与高分辨气相色谱-高分辨质谱法(HRGC-HRMS)测定值吻合度高,检测结果误差<20%。本方法具有操作简便、快速、检测结果可靠等特点,适用于基层检测机构对牛奶中二噁英的快速筛查。     

Determination of multiresidue of avermectins in bovine liver by an indirect competitive ELISA

An ELISA was developed to detect multiresidues of avermectins (AVMs) including abamectin (ABM), ivermectin (IVM), and eprinomectin (EPR) in bovine liver. The modified ABM, 4'-O-succinoyl-ABM was conjugated to bovine serum albumin as the immunogen for the preparation of polyclonal antibodies to AVMs and conjugated to ovalbumin as the coating antigen for the ELISA. Serum with the highest antibody titers to AVMs, which had a cross-reactivity of 100% with ABM, 145.4% with EPR, and 25% for IVM, was selected for the development of an indirect competitive ELISA. The ELISA could detect ABM, IVM, and EPR residues in bovine liver tissues, with a limit of quantitation of 1.06 ng/mL for all three AVMs. Optimal pH, ion strength, organic solvent, and duration of incubations were investigated to increase the sensitivity of the ELISA. Recoveries of these drugs ranged from 53.8% to 80.6% with inter-assay coefficients of variation (CV) of 3.4-17.9% and intra-assay CV of 5.5-14.7%. Analysis results of field samples by the ELISA were consistent with those by a previously developed HPLC method. The ELISA can be used as a rapid method for screening of AVMs residues in bovine liver.     

Antioxidative activity of urate in bovine milk

The antioxidative effects of urate on peroxidase-induced protein oxidation and light-induced riboflavin degradation and lipid oxidation in whole milk were studied. In addition, experiments using ascorbate were conducted to directly compare the antioxidative activity of urate and ascorbate. The presence of urate and/or ascorbate (10-30 mg/L) lowered peroxidase-induced formation of dityrosine by 44-96% in unpasteurized whole milk. No synergistic effect of urate and ascorbate on peroxidase-induced dityrosine formation was registered, but merely an additive effect. Light exposure of pasteurized whole milk showed that ascorbate was oxidized at the expense of urate, which indicated ascorbate-mediated recycling of the urate radical. Moreover, both urate and ascorbate (30 mg/L) retarded light-induced lipid oxidation in pasteurized whole milk as measured by formation of lipid hydroperoxides with urate being the most effective (28% reduction in lipid hydroperoxides) compared with ascorbate (14%). Finally, addition of urate or ascorbate (300 mg/L) to pasteurized whole milk showed a slight protective effect against light-induced degradation of riboflavin with urate being the most effective.     

Liquid chromatographic determination of cephapirin residues in milk

A liquid chromatographic (LC) method was developed for quantitative determination of cephapirin residues in milk that also resolved cephapirin from ampicillin, cloxacillin, and penicillin G. Diluted milk was passed through a C18 cartridge on which the cephapirin was adsorbed; then, interfering material was removed by washing with water and methylene chloride and cephapirin residues were eluted with methanol-acetonitrile (25 + 75). After drying, residues were dissolved in the mobile phase for injection. The LC system had an ultrasphere-ODS column with RP-18 Spheri-10 guard column and a UV detector with a 254 nm filter. The mobile phase was 85% sodium acetate (0.01M) and 15% methanol-acetonitrile (25 + 75) with a flow rate of 1 mL/min. Sensitivity was 20 ppb or less with a recovery of 61-80% in the range studied. Other beta-lactam antibiotics tested did not interfere with detection of cephapirin. Analysis of 30 samples of commercial homogenized milk obtained for a survey of antibiotics in consumer milk in Canada revealed no detectable cephapirin residues.     

Determination of florfenicol amine residues in animal edible tissues by an indirect competitive ELISA

Florfenicol (FF) is a broad-spectrum antibiotic used increasingly in aquaculture, livestock, and poultry to treat diseases. To avoid using labor-intensive instrumental methods to detect residues of FF in food and food products, a simple and convenient indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method for florfenicol's major metabolite, florfenicol amine (FFA), was developed using a polyclonal antibody prepared in this study. FFA was covalently attached to carrier protein as immunogen by using the glutaraldehyde method. The antibodies obtained were characterized by an ELISA method and showed excellent specificity and sensitivity with the 50% inhibition values (IC 50) of 3.34 microg/L for FFA in PBS buffer. In the ELISA, sample extractions were performed by ethyl acetate/ammonium hydroxide (90 + 10, v/v) following combined acid hydrolysis of FF and its known metabolites. The limits of detection (LOD) calculated from the analysis of 20 known negative swine muscle, chicken muscle, and fish samples were 3.08, 3.3, and 3.86 microg/kg (mean + 3 SD), respectively. Recoveries of FFA fortified at the levels of 5, 50, 100, and 300 microg/kg ranged from 64.6 to 124.7%, with coefficients of variation of 11.3-25.8% over the range of FFA concentrations studied. Validation of the ELISA method with FFA-fortified swine muscle at the levels of 10, 50, 100, and 200 microg/kg was carried out using GC, resulting in a similar correlation in swine muscle ( r = 0.97). The results suggest that this ELISA is a specific, accurate, and sensitive method, which is suitable for use as a screening method to detect residues of FFA in animal edible tissues.     

Polyclonal-antibody-based ELISA to detect milk alkaline phosphatase

Polyclonal antibodies (PAb) prepared against bovine milk alkaline phosphatase (ALP) were used to develop a competitive indirect (CI) ELISA. Anti-ALP PAb were specific for milk ALP and did not react with ALP from E. coli or bovine and calf intestinal mucosa. Anti-ALP PAb were 20% cross-reactive with bovine placenta ALP. The anti-ALP antibodies also did not recognize bovine serum albumin, acid glycoprotein, ovalbumin, ferritin, and casein, although some cross-reactivity was observed with whey protein isolate. Anti-ALP PAbs reacted with deglycosylated native ALP, but did not recognize ALP denatured at 100 degrees C in 2% SDS or deglycosylated denatured ALP. When buffered solutions of milk ALP were heated at 70 degrees C, ALP activity decreased at a faster rate than ALP content determined by CI-ELISA. The ELISA differentiated between native and heat denatured ALP. Further studies are warranted to determine if an ELISA can be used to verify pasteurization of fluid milk.     

Determination of depletion and statistical distribution of morantel-related residues in bovine milk following administration of morantel tartrate to dairy cows

Residue depletion studies were conducted in dairy cattle to monitor morantel-related residues in milk following oral administration of morantel tartrate (Rumate. Eleven lactating cows of various ages, periods of lactation, and known milk production were orally dosed with the bolus formulation of morantel tartrate with an actual dose range of 8.4-9.8 mg/kg body weight. Representative samples of milk were collected at 10-14 h intervals post-dose, and subsamples were assayed for the major and minor hydrolysis products of morantel-related residues, MAPA and CP-20,107. Residues assayed as precursors of MAPA peaked at the second milking (24 h post-dose) and were below 25 ppb (range: less than 12-24 ppb). Precursors of CP-20,107, which confirm the identity of morantel, also peaked at 24 h post-dose (range: 2.1-3.3 ppb) and declined rapidly thereafter. A statistical model was used to project the level of residues at the upper limit of 99% of the total target animal (i.e., dairy cattle) population with 95% confidence. The calculated peak levels from this model were 50 and 5.0 ppb for morantel-related residues convertible to MAPA and CP-20,107, respectively.     

Organophosphorus pesticide residues in Mexican commercial pasteurized milk

A study was conducted to measure residues of 13 organophosphorus (OP) pesticides, widely used as dairy cattle ectoparasiticides or in crops used for animal feed, in homogenized and pasteurized Mexican milk samples. Four different milk brands with high distribution were collected biweekly during a 12 month period (n = 96) in supermarkets. OP pesticide residues were measured by gas chromatography with a flame photometric detector. Approximately 39.6% of the samples contained detectable levels of OP pesticide residues. Eight samples contained residues exceeding established maximum residue limits (MRL), and the OP pesticides present in these samples were dichlorvos (five samples), phorate, chlorpyrifos, and chlorfenvinphos (one sample, respectively). Average residues of 13 OP pesticides measured were below established MRLs ranging between 0.0051 and 0.0203 ppm.     

Disposition of doramectin milk residues in lactating dairy sheep

Doramectin (DRM) is a broad spectrum macrocyclic lactone antiparasitic drug not approved for use in dairy animals. However, DRM and other endectocide compounds are widely used extra-label to control endo- and ectoparasites in dairy sheep. The plasma disposition kinetics and the pattern of DRM excretion in milk were characterized following its subcutaneous administration to lactating dairy sheep. DRM concentration profiles were measured in plasma and milk samples after validation of a specific HPLC-based methodology. DRM was detected between 1 h and 30 days post-treatment. DRM concentrations of 0.48 ng.mL(-1) (plasma) and 1.03 ng.mL(-1) (milk) were measured at 30 days post-treatment. DRM was extensively distributed from the bloodstream to the mammary gland, and large concentrations were excreted in milk. The peak concentrations and total amount of DRM recovered in milk (expressed as area under the concentration versus time curve) were 3-fold higher than those measured in plasma; 2.44% of the total DRM dose was excreted in milk. The long persistence of DRM milk residues should be seriously considered before its extra-label use in dairy animals is recommended.     

Determination of novobiocin residues in milk, blood, and tissues by liquid chromatography

Residues of novobiocin in milk, blood, and tissues can be detected by microbiological tests but cannot be distinguished from other antibiotics. A simple liquid chromatographic (LC) method was developed for identification of residues. Tissues were blended and milk and blood serum were mixed with 0.2M NH4H2PO4. The mixture was deproteinized by adding aqueous methanol and filtering. The LC apparatus consisted of a variable wavelength detector, set at 340 nm, an automatic loop injector, and a C18 column with guard cartridge. The flow rate was 1 mL/min and the solvent mixture of 0.01M H3PO4-acetonitrile-methanol was programmed from 50 + 0 + 50 (0-1 min) to 20 + 80 + 0 (20 min). Novobiocin was concentrated directly by solid-phase extraction on the analytical column. Five or more 200 microL aliquots of the filtrate in water-methanol (1 + 1) (adjusted if necessary) were injected with the column solvent at 50 + 0 + 50. After the final injection, the program was run to completion. Recoveries were 90-100% with sensitivities of 0.05 ppm or less. The procedure should be adaptable for use with formulations and feeds.     

ELISA to detect proteolysis of ultrahigh-temperature milk upon storage

Casein proteolysis can occur in milk during storage leading to its gelation. The two main proteolytic systems suspected to be involved are the plasmin and the proteases produced by psychrotrophic bacteria. The latter have been shown to cleave kappa-casein at the Phe105-Met106 bond. Although several techniques allow the determination of plasmin in milk, few rapid and easy-to-perform analytical techniques are available to check for bacterial proteolytic activity. This study presents the development of an inhibition ELISA allowing for the quantification of the kappa-casein intact at the Phe105-Met106 bond. It uses a monoclonal antibody specifically directed against this peptide bond that binds to the protein as long as the molecule's cleavage site is intact but not when it is cleaved. This simple technique allows for the rapid analysis of more than 20 samples within 3 h. Applied to commercial milks, this assay allowed for the detection of unstable milk.