Determination of Oxytetracycline, Tetracycline, and Chlortetracycline in Milk by Liquid Chromatography with Postcolumn Derivatization and Fluorescence Detection (original) (raw)
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Trace analysis of oxytetracycline and tetracycline in milk by high-performance liquid chromatography
Journal of Agricultural and Food Chemistry, 1990
A high-performance liquid chromatographic (HPLC) method for the determination of oxytetracycline (OTC) and tetracycline (TC) residues in milk a t levels as low as 10 ppb has been developed. Milk was acidified a t pH 2.7 and extracted with acetonitrile. The extract was partly purified by treatment with ammonium sulfate solution and concentrated into a phosphate buffer, pH 8.2. Following addition of tetrabutylammonium reagent (TBA), tetracyclines were extracted as ion pairs with TBA into dichloromethane, reextracted into acid, and analyzed on a reversed-phase CIS, 5 pm, column. Overall recovery was found to be 72.7 f 1.2% for OTC and 85.1 f 1.3% for TC. The linearity was excellent for both compounds in the range examined (r = 0.9996, 23.7-190 ppb of OTC in milk; r = 0.09995, 26.5-212 ppb of TC in milk). Precision data based on within-day and among-days variation suggested an overall relative standard deviation of 5% for OTC and 4.2% for TC.
Simultaneous determination of tetracycline, oxytetracycline, and 4-epitetracycline in milk
A reversed-phase high-performance liquid chromatography with photodiode-array detection (HPLC-PAD) was optimized and validated for the simultaneous determination of tetracycline (TC), 4-epitetracycline (4-epiTC) and oxytetracycline (OTC) in milk. Milk samples were extracted and cleaned-up using solid-phase extraction Discovery SPE DSC-18 tubes. The separation were accomplished in less than 8 min in a Waters Symmetry C18 column at ambient temperature with a mobile phase consisted of 0.010 M aqueous oxalic acid:acetonitrile:methanol (150:20:20 by volume). Quantitation was carried out by the peak area method, with detection limits of 2.0 lg/l of each tetracycline. Average recoveries of TC, 4-epiTC and OTC from spiked samples at the four concentrations (0.25, 0.5, 1.0 and 1.5 lg/ml) were 91.5, 71.5 and 83.1, respectively, with their standard deviations less than 4% within a day and 7% between days. This method was applied for the simultaneous determination of TC, 4-epiTC and OTC in market milk samples purchased from local supermarkets. Oxytetracycline was found being present in all samples in a concentration range of 13-106 lg/l, 4-epiTC in most samples at 18-65 lg/l, TC in one sample at 44 lg/l.
Iranian Journal of Veterinary Medicine, 2014
BACKGROUND: Tetracyclines (TCs) are broad-spectrumantibiotics that are widely used in veterinary medicine. Thepresence of TCs residues in milk is a public health concern allover the world. OBJECTIVES:This study aimed to determine TCsresiduals in pasteurized milk marketed by some dairy companiesin Tehran from April 2011 to March 2012. METHODS: 432pasteurized milk samples were purchased from supermarketssupplying the milk products of 12 major dairy companies inTehran (3 samples from each company every month), and theywere stored at -20 0C until analysis. Oxytetracycline (OTC) andTetracycline (TC) residues in each sample were extracted by aliquid - liquid phase procedure and quantitated using a highperformance liquid chromatographic (HPLC) method. Chromatographicconditions included a mobile phase as oxalic acidbuffer- acetonitril (80: 20) with a flow rate of 1mL/min and UVdetectionat 355 nm. RESULTS: TCs residuals in most milksamples were lower than 100 ppb, maximum residue level(MRL);...
Monitoring of oxytetracycline in ovine milk by high-performance liquid chromatography
Journal of Pharmaceutical and Biomedical Analysis, 1999
A method for 'in vivo' determination of the oxytetracyclin residues in ovine milk at low levels is described. Two groups of Sardinian breed sheep were treated with a dose of oxytetracycline by intramammary infusion and intramuscular administration, respectively. Oxytetracycline residues in extracts obtained from a preliminary cleanup procedure, were detected by an isocratic high-performance liquid chromatography (HPLC) method. Linear calibration plots were obtained over a large concentration range of 1 mg ml − 1 -10 ng ml − 1 , with correlation coefficients higher than 0.996. Recoveries between 85.8 and 98.9% were obtained. Limit of detection (LOD) and limit of determination (LOQ) were 5.2 and 17.5 ng ml − 1 , respectively. This method would be useful for routine monitoring of oxytetracycline residues in ovine dairy milk. : S 0 7 3 1 -7 0 8 5 ( 9 9 ) 0 0 0 5 0 -3
Testing of Raw Milk for Tetracycline Residues
Journal of Dairy Science, 1998
A newly improved Bacillus calidolactis tube diffusion test and two postscreening test systems-a receptor assay (Charm HVS; Charm Sciences, Inc., Malden, MA) and a newly developed Bacillus cereus ATCC 11778 mycoides test system-were evaluated for the detection and identification of tetracycline residues using 973 samples of bulk milk taken at random in The Netherlands. All milk samples were assayed with the B. calidolactis tube and the receptor test. The milk samples testing as suspect or positive with one or both of the test systems were analyzed with HPLC (limit of detection, 10 ng/ml) and the recently developed B. cereus test system. The B. calidolactis tube diffusion test detected tetracycline residues >45 ng/ml in milk. With the B. cereus test plate, residues of oxytetracycline and tetracycline of >30 ng/ml milk were detected; for chlortetracycline and doxycycline, the detection limit was 10 ng/ml. Raw milk exhibiting inhibition diameters of <20 mm on the B. cereus test plate fulfilled the European Union criterion for maximum residue level for tetracyclines of <100 ng/ml (including their 4-epimer derivatives). The detection limits of the receptor assay depended on the type of milk used. The scintillation counts that were obtained for control samples of bulk milk were considerably lower than for the milks obtained from Charm Sciences, Inc. or processed using UHT pasteurization. One of 973 milk samples was suspect for tetracycline residues by means of the B. calidolactis tube test as well as by the receptor assay; 8 other samples were also considered to be positive using the receptor assay alone. The presence of tetracycline residues could not be proved for these 9 samples (residue concentration, <10 ng/ml) with HPLC. We concluded that the receptor assay was not reliable to detect tetracycline residues in raw milk at <150 ng/ml. The B. cereus test plate was determined to be an inexpensive, reliable alternative for the receptor assay for detection of tetracycline residues.
Highly Sensitive Immunochromatographic Identification of Tetracycline Antibiotics in Milk
International Journal of Analytical Chemistry, 2015
A rapid immunochromatographic assay was developed for the control of tetracycline (TC). The assay is based on the competition between immobilized TC-protein conjugate and TC in a tested sample for binding with polyclonal anti-TC antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. Conjugation of colloidal gold and the total immunoglobulin (IgG) fraction of polyclonal antibodies was used to increase the assay sensitivity to ensure low content of specific antibodies in the conjugate. This allowed effective inhibition of free TC and conjugate binding in the strip test zone. Photometric marker registration allows control of the reduction of binding, thereby enhancing detection sensitivity. The proposed assay allows TC to be detected at concentrations up to 20 ng/mL, exceeding the limit of detection of the known analogues, in a wide working range (more than two orders) of 60 pg/mL to 10 ng/mL, ensured through the use of polyclonal antibodies. The assay time is 10 min. The efficiency of the designed assay is shown to identify TC in milk; the degree of recovery of TC ranges from 90 to 112%. The precision of the concentrations measurements was no more than 10%.
In-house validation for multi-residue analysis of tetracycline in cow milk by HPLC with UV detection
Semina: Ciências Agrárias, 2017
The indiscriminate use of antibiotics in dairy cattle without complying with the waiting period results in residual contamination, whose effective control in produced milk requires validated methods toensure analytical results. The aim of this study was to optimize and validate the HPLC-UV/VIS method at 365 nm for analyzingthe tetracycline in pasteurized cow milk in accordance with the European Community (2002/657/EC). Spiked milk with analytes (oxytetracycline, tetracycline, doxycycline, and chlortetracycline) was submitted to deproteinization and cleaning by a C18 solid-phase column and analyzed by HPLC using a gradient system with 0.01 mol L?1 oxalic acid-acetonitrile-triethylamine (90:9.9:0.1) and acetonitrile on a reverse phase (C18) column. Accuracy and precision were assessed by adding analytes to levels of 0.5, 1, and 1.5 times the permissible maximum limit allowed in Brazil. The method presented selectivity with a decision limit (CC?) and detection capability (CC?) ranging ...
Asian Journal of Dairy and Food Research, Volume 43 Issue 2: 306-312 (June), 2024
Background: This study aimed to identify tetracycline residues in milk consumed in the region of Constantine (northeast Algeria) using ELISA based tetracyclin kit. Methods: A total of 180 samples were analyzed (fresh cow milk and imported powdered milk). To compare ELISA and HPLC detection values, 22 fresh milk ELISA positive samples were confirmed by HPLC analysis. Result: 92.5% of fresh milk samples contained tetracycline residues at values between 5 and 74 µg/L and 33.3% of the samples showed concentrations between 49 and 74 µg/L, that exceed the MRLs recommended by the FDA. No significant differences (p>0.01) were found between the values obtained by the two methods.
Analysis of residual oxytetracycline in fresh milk using polymer reversed-phase column
Food Chemistry, 2008
A simple and rapid reversed phase high performance liquid chromatograph (HPLC) method for analysis of oxytetracycline (OTC) was developed and applied in the determination of the antibiotic in fresh milk sample. Isocratic elution was performed with acetic acid:water (pH 4.5):acetonitrile (4:68:28), using a polymer reversed-phase (PLRP) column and UV detection at 354 nm wavelength. The average recoveries of OTC spiked milk at 0.1, 0.2, 0.5 and 100 ng/mL were in excess of 92% with intraday and interday precision between 0.8% and 6.6% respectively. A good linearity was established between the range 100-1000 ng/mL with r 2 = 0.9995. The limit of detection and quantitation were 30 and 100 ng/mL respectively. The method demonstrated successful application for analysis of 100 milk samples. Two samples out of 70 from livestock keepers tested OTC positive while none of the 30 samples from milk centers tested positive.
A Group-Specific Microbiological Test for the Detection of Tetracycline Residues in Raw Milk
Journal of Agricultural and Food Chemistry, 2000
The potentiality of using a luminescent Escherichia coli strain for the specific detection of tetracycline residues in raw bovine milk was investigated. The sensor cells contain a reporter plasmid carrying the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive control region from transposon Tn10. Incubation of the cells with the sample containing tetracyclines increases the light emission of the sensor cells. The most sensitive tetracycline detection was achieved in 120 min and by using CDTA as a chelating agent in the assay. Heat-treatment of milk before the assay decreased the variations in background luminescence signals and in tetracycline-induced luminescence between different milk samples. The detection limits for tetracycline, oxytetracycline, chlortetracycline, doxycycline, methacycline, demeclocycline, and minocycline were between 2 and 35 ng/mL. Nontetracycline antibiotics did not significantly interfere with the detection of tetracyclines.