A new electrochemical enzyme-linked immunosorbent assay for the screening of macrolide antibiotic residues in bovine meat.

A new sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed, using the mouse monoclonal antibodies anti-erythromycin and anti-tylosin. The competitive indirect assay was performed using an erythromycin (or tylosin)-BSA conjugate as a coating molecule; after competition between free and coated analytes for the antibodies, the activity of the horseradish peroxidase-labelled antiglobulins was measured electrochemically using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate. The detection limit of the assay was 0.4 ng ml(-1) for erythromycin and 4.0 ng ml(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.4 ng ml(-1) for erythromycin and 13.0 ng ml(-1) for tylosin. The specificity of the assay was assessed by studying the cross-reactivity of various macrolides other than erythromycin and tylosin. The results indicate that the monoclonal antibodies anti-erythromycin and anti-tylosin can readily distinguish the target compound from other macrolides, with the exception of roxithromycin, a semisynthetic macrolide antibiotic derived from erythromycin. Fortified and real samples were analysed by the developed ELISA method and results confirmed by micro-LC-MS-MS using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest. The ELISA assay showed precision (RSD) values ranging from 6.3 to 11.4% for erythromycin and from 7.5 to 12.6% for tylosin; the accuracy (relative error, RE) ranged from -16.0 to -9.8% and from -9.5 to 8.0% for erythromycin and tylosin, respectively. All results obtained demonstrate that the electrochemical ELISA is a suitable method for a sensitive, simple, rapid and reliable screening of the two macrolides in animal tissues.     

Confirmatory method for macrolide residues in bovine tissues by micro-liquid chromatography-tandem mass spectrometry

A new confirmatory method for three macrolides (tylosin, tilmicosin and erythromycin) in bovine muscle, liver and kidney by micro-LC-MS-MS using an atmospheric pressure ionisation source and an ionspray interface has been developed. Roxithromycin was used as internal standard. The molecular related ions, [M+2H]2+, at m/z 435 for tilmicosin, and [M+H]+, at m/z 734 and 916 for erythromycin and tylosin, respectively, were the precursor ions for collision-induced-dissociation and two diagnostic product ions for each macrolide were identified for the unambiguous confirmation by selected reaction monitoring LC-MS-MS. Precision values (relative standard deviations) were all below 14.9%, whereas the overall accuracy (relative error) ranged from -17.7 to -9.8% for tylosin, from -17.5 to -10.7% for tilmicosin and from -19.6 to -13.7% for erythromycin, in all the investigated bovine tissues. The limits of quantification were 30 (muscle) or 40 (liver, kidney) microg kg(-1), 20 (muscle) or 150 (liver, kidney) microg kg(-1), 50 (muscle, liver) or 80 (kidney) microg kg(-1), 20 (muscle, liver) or 50 (kidney) microg kg(-1) for tylosin, tilmicosin, erytromycin and roxithromycin, respectively.     

Development of a highly sensitive enzyme-linked immunosorbent assay for bisphenol A in serum

4,4'-Isopropylidenediphenol, bisphenol A (BPA), was derivatized to BPA-carboxymethylether (BPA-CME), BPA-carboxypropylether (BPA-CPE) and BPA-carboxybutylether (BPA-CBE), and then linked to bovine serum albumin (BSA). The BPA-BSA conjugates were injected into female New Zealand White rabbits, which then generated six kinds of polyclonal antibodies. In addition, BPA and bisphenol B (BPB)-enzyme conjugates were derivatized to BPA-CME, BPA-CPE, BPA-CBE, BPA-carboxyphenylether (CPhE) and BPB-CPE, and then linked to horseradish peroxidase (HRP). An enzyme-linked immunosorbent assay (ELISA) was developed and the specificity of the antibodies was confirmed by comparison with pre-immune serum and by competitive assays using different dilutions of BPA standards. Although anti-BPA antibodies cross-reacted with BPB by more than 13.6% at all dilutions used, cross-reaction with phthalates and phenols occurred only less than 0.1%. The combination with the highest sensitivity was obtained using anti-BPA-CME-BSA antibody and BPA-CPhE-HRP conjugate. ELISA successfully detected BPA in human serum at concentrations as low as 0.3 ng mL(-1), and over a measurable range of 0.3-100 ng mL(-1). Recovery tests were carried out by adding BPA to three kinds of human serum, and ranged from 89.7 to 97.3%, from 85.4 to 94.9% and from 81.9 to 97.4%, respectively. The correlation between the results from ELISA and gas chromatography-mass spectrometry (GC-MS) for BPA in spiked serum was r2 = 0.990, indicating that the proposed method is a potential tool for screening a large number of human serum samples.     

Antibody-biotemplated HgS nanoparticles: extremely sensitive labels for atomic fluorescence spectrometric immunoassay

In this work, antibody goat anti-human IgG as a scaffold was employed for the synthesis and biofunctionalization of HgS nanoparticles (NPs) via a facile one-pot process. After a complete sandwich-type immunoreaction among primary antibody, human IgG and secondary antibody labeled with HgS NPs, a large number of mercury ions released from captured HgS NPs dissolution were quantitatively detected by chemical vapor generation atomic fluorescence spectrometry (CVG-AFS). Taking advantage of the signal amplification property of HgS NPs and the high sensitivity of CVG-AFS, the assay detected human IgG with a limit of detection (S/N = 3) of 0.6 ng mL(-1) (4.0 fmol mL(-1) or 0.4 fmol) and the response was linear over a dynamic range from 1.0 to 5.0 × 10(4) ng mL(-1) with a correlation coefficient of 0.996. A relative standard deviation (RSD) of 1.0 × 10(2) ng mL(-1) human IgG was 1.5% for within-batch (intra-assay) and 4.5% for between-batch (inter-assay). Other proteins, such as goat anti-rabbit IgG, goat anti-human IgG, rabbit anti-human IgG, carcinoembryonic (CEA), α-fetoprotein (AFP), human serum albumin (HSA) and bovine serum albumin (BSA) did not significantly interfere with the assay for human IgG. The analytical result of HgS NPs with AFS-based immunoassay technology for the quantification of human IgG in human serum from patients is in good agreement with the result obtained by conventional immunoturbidimetric method. The consequence shows that the novel immunosensor possessed satisfactory precision, extremely high sensitivity, high selectivity and could be applied for the quantification analysis of real samples.     

Multiresidue determination of zeranol and related compounds in bovine muscle by gas chromatography/mass spectrometry with immunoaffinity cleanup

A gas chromatography/mass spectrometry (GC/MS) method with immunoaffinity cleanup was developed for the determination of zeranol and related compounds, taleranol, zearalanone, and alpha-zearalenol in bovine muscle. Muscle samples were extracted with methanol and cleaned up with immunoaffinity chromatography (IAC) columns containing monoclonal antibodies raised against zeranol coupled to CNBr-activated Sepharose 4B. After derivatization, the compounds were analyzed by GC/MS. The dynamic column capacities for zeranol, taleranol, zearalanone, and alpha-zearalenol were 2639.7, 2840.3, 2731.5, and 2736.3 ng/mL Sepharose gel, respectively. The limits of detection and quantification were 0.5 and 1.0 ng/g, respectively, for all 4 compounds. Mean recoveries were 79.6-110.7% with coefficients of variation of 3.2-11.4% at spiked levels of 1.0-5.0 ng/g. This IAC-GC/MS method may be used for the determination of zeranol, taleranol, zearalanone, and alpha-zearalenol residues in bovine muscle, and possibly other tissues.     

Development of DNA-Designed Avian IgY Antibodies for Quantitative Determination of Bovine Interferon-Gamma

Interferon-gamma (IFN-γ), a cytokine produced by sensitized T lymphocytes, is one of the key elements in defining T helper 1 lymphocyte immune responses. Quantitative evaluation of IFN-γ expression could provide an important analytical tool for measurement of cell-mediated immunity and investigating immune responses to infectious diseases. Method of DNA-designed avian IgY antibodies was used for production of monospecific polyclonal antibodies that allows quantification of the recombinant bovine IFN-γ protein. IFN-γ cDNA was subcloned and expressed in mammalian expression plasmid (pcDNA3.1(+)) under the control of the human cytomegalovirus promoter. Chickens were immunized by plasmid DNA, and eggyolk antibodies extracted from eggs were collected after immunization. IgY-specific antibodies were evaluated by an antigen capture enzyme-linked immunosorbent assay (ELISA) using recombinant IFN-γ. Based on the results, developed bovine IFN-γ capture ELISA could detect up to 1 ng/ml of IFN-γ by 64-fold diluted IgY. Monospecific anti-bovine IFN-γ antibodies generated in chickens are useful for quantifying different concentrations of recombinant bovine IFN-γ, which is expressed in cell culture.     

Development of an enzyme-linked immuno-sorbent assay (ELISA) method for carbofuran residues

The haptens 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-amino]butanoic acid (BFNB) and 6-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)-carbonylamino]hexanoic acid (BFNH) were synthesized and then used to develop a rapid,specific and sensitive ELISA method to determine residues of the pesticide carbofuran in a variety of matrices. A hybridoma cell line (5D3) producing anti-carbofuran monoclonal antibodies (MAbs) was also established. Based on the MAbs in combination with the heterologous hapten BFNH coupled to either horseradish peroxidase (HRP) or ovalbumin(OVA), four ELISAs (formats I-IV) for the quantification of carbofuran were developed and compared. Among them, the optimized format II (the conjugate-coated direct competitive ELISA) showed the best characteristics, with an IC50 value of 18.49 ng/mL, a limit of detection of 0.11 ng/mL and the shortest assay time (1 h). This ELISA method was then applied to the determinations of carbofuran in environmental water, soil and food samples. The relative standard deviations (R.S.D.s) ranged from 1.8% to 21.3% and the mean recoveries were 104.6%, 108.3%, 106.3% and 100.1% for water, soil, lettuce and cabbage, respectively. Thus, the ELISA method of format II exhibited the potential to develop commercial ELISA kits for a rapid detection of carbofuran for human health and environmental safety.     

Synthesis of haptens of organophosphorus pesticides and development of immunoassays for fenitrothion

A simple synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (aminocarboxylic acid) attached at the pesticide thiophosphate group was developed. While the previous synthetic approach for this type of haptens requires seven steps, the present method involves only two steps. Using this method, five haptens of fenitrothion were synthesized and two of them were conjugated to proteins to be used as immunogens for production of polyclonal antibodies. Using the antibodies and a coating antigen, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for fenitrothion was developed, which showed an IC₅₀ of 3.7 ng/mL with a detection limit of 0.5 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 5.6 ng/mL with a detection limit of 0.4 ng/mL. The antibodies in both assays showed negligible cross-reactivity with other OP pesticides except with the insecticide parathion-methyl only in the direct ELISA. Recoveries of fenitrothion from fortified lettuce and rice samples ranged 84-116 and 100-121%, respectively.     

Optimization of the experimental conditions for macrolide antibiotics in high performance liquid chromatography by using response surface methodology and determination of tylosin in milk samples

The simple, rapid and sensitive liquid chromatographic separation of five macrolides (tilmicosin, erythromycin, tylosin, roxithromycin and josamycin) widely used in food producing animals was developed. Response surface methodology was used as an optimization method of mobile phase, column temperature and pH to provide the best resolution of these analytes. The separation was performed by using an end-capped X-Terra RP-18 column (250 × 4.6 mm I.D × 5 m) with an isocratic system of 15 mM hydrochloric acid (pH 2.5)-acetonitrile as the mobile phase at a temperature of 30°C and flow-rate of 1.0 mL/min. The suitability of the method for multi-residue determination of the macrolides is demonstrated by the analysis of milk samples spiked with tylosin. Roxithromycin was used as internal standard. The recovery of tylosin was quite good as 90.8%. The limit of quantification and detection limit were 0.024 and 0.007 μg/mL, respectively. The method was successfully applied to determination of macrolides at levels below the maximum concentration legally allowed in milk samples.     

Immunochemical-based method for detection of hazelnut proteins in processed foods

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.     

Simultaneous analysis of twenty-one glucocorticoids in equine plasma by liquid chromatography/tandem mass spectrometry

A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for screening for that drug. Analyte confirmation was performed by using the three most intense product ions formed from the precursor ion and the corresponding mass ratios. The recovery of the 21 GCC when spiked into blank plasma at 5 ng/mL was 45-200% with coefficient of variation (CV) from 0.3-18%. The limit of detection (LOD) and that of quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively, whereas that of confirmation (LOC) was 100-300 pg/mL depending on the analyte. Intra- and inter-day precisions expressed as CV for quantification of 1 and 10 ng/mL was 1.0-17%, and 0.51-19%, respectively, and the accuracy was from 84-110%. The linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997). Estimated measurement uncertainty was from 11-37%. This study was undertaken to develop a method for simultaneous screening, identification, quantification and confirmation of these agents in post-race equine plasma samples. The method has been successfully applied to screening of a large number of plasma samples obtained from racehorses in competition and in pharmacokinetic studies of dexamethasone in the horse and concurrent changes in endogenous GCC, hydrocortisone and cortisone. The method is simple, sensitive, selective and reliably reproducible.     

Determination of macrolide antibiotics in porcine and bovine urine by high-performance liquid chromatography coupled to coulometric detection

A high-performance liquid chromatography method coupled to coulometric detection has been applied for the determination, in a single run, of up to eight macrolide antibiotics (erythromycin [ERY], tylosin [TYL], tilmicosin [TILM], spiramycin 2 [SPI 2], spiramycin 3 [SPI 3], josamycin [JOS], kitasamycin [KIT], and rosamicin [ROS]) in spiked porcine and bovine urine. Quantification was performed using matrix-matched calibration with roxithromycin (ROX) as the internal standard. The detection limits for each drug were below 3.5 ng injected (equivalent to an initial concentration below 0.07 mg L^–1) for porcine urine and below 5 ng injected (equivalent to an initial concentration below 0.10 mg L^–1) for bovine urine. Recoveries from urine samples spiked at three different concentrations within the linear range were not significantly dependent on concentration. The entire procedure provides average macrolide recoveries ranging from 69.7 to 96.6% for bovine urine and from 75.5 and 96.1% for porcine urine.     

A sensitive and selective enzyme-linked immunosorbent assay for the analysis of Para red in foods

Para red is a synthetic dye and a potential genotoxic carcinogen. A hapten mimicking Para red structure was synthesized by introducing a carboxyl to the naphthol part of Para red and coupled to carrier protein to form an immunogen for the production of specific antibodies. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Para red in food samples. The limit of detection and inhibition half-maximum concentrations of Para red in phosphate buffered saline with 10% methanol were 0.06 and 2.2 ng mL(-1), respectively. Cross-reactivity values of the ELISA with the tested compounds including Sudan red I, II, III, IV, and G, sunset yellow, 2-naphthol, and 4-nitroaniline were ≤0.2%. This assay was used to determine Para red in tomato sauce, chilli sauce, chilli powder and sausage samples after ultrasonic extraction, cleanup and concentration steps. The average recoveries, repeatability (intraday extractions and analysis), and intra-laboratory reproducibility (interday extractions and analysis) were in the range 90-108%, 4-12% and 8-17%, respectively. This assay was compared to a high-performance liquid chromatographic method for 28 samples, displaying a good correlation (R(2) = 0.95). Para red residues in 53 real world samples determined by ELISA were below the limit of detection.     

Innovative mixture of salts in the quick,easy, cheap,effective, rugged,and safe method for the extraction of residual macrolides in milk followed by analysis with liquid chromatography and tandem mass spectrometry

A simple extraction technique has been developed for seven macrolide antibiotics in milk. The procedure involves a modified quick, easy, cheap, effective, rugged, and safe method based on acetonitrile extraction, followed by the addition of a mixture of salts (sodium sulfate, sodium chloride, and potassium carbonate) not yet reported in literature. The method was validated for tylosin and was selective, free of matrix effect, and linear in the range of 0.78–18.75 ng/mL in the final extract, corresponding to 0.125–3 times the maximum residue limit. The limit of detection, limit of quantification, decision limit, and detection capability were, respectively, 0.84, 2.79, 58.4, and 71.7 μg/kg. The overall average recovery at 25, 50, and 75 μg/kg ranged from 89–97%. Repeatability and intermediate precision expressed by relative standard deviations were below 10.5 and 12%, respectively. The extension of the validation for spiramycin, throleandomycin, oleandomycin, roxithromycin, erythromycin, and clarithromycin is under consideration since the procedure proved to be able to efficiently extract all studied macrolides, with recoveries from 74–104% at 50 μg/kg for tylosin, erythromycin, spiramycin, and oleandomycin and 20 μg/kg for throleandomycin, roxithromycin, and clarithromycin.     

Quantitation ofE. coli protein impurities in recombinant human interferon-γ

A multiple antigen ELISA for E. coli proteins (ECPs) that may be present in purified recombinant human interferon-gamma (rIFN-gamma) was developed. SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In spike recovery studies, rIFN-gamma at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50%. To determine ECP content in purified rIFN-gamma, 0.2 mg/mL of rIFN-gamma was added to the standard curve diluent to compensate for enhanced immunoreactivity. The assay was precise (interassay precision of ECP controls < or = 4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-gamma (1 mg/mL). Linearity of dilution for ECPs spiked into rIFN-gamma was obtained (r = 0.999). Moreover, linearity of dilution was obtained for ECPs in "in-process" samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA. ECPs were not detectable in several purified lots of rIFN-gamma. Therefore, these lots contained < 1.3 ppm ECPs.     

Determination of levofloxacin (the Levorotatory Stereoisomer of Ofloxacin) in milk by an indirect enzyme-linked immunosorbent assay

In this work we obtained polyclonal antibodies for levofloxacin. We synthesized conjugates of levofloxacin with cationized BSA for immunization and with ovalbumin for development of an ELISA for the detection of levofloxacin. The method is characterized by a range of detectable concentrations of 0.03 ng/mL to 0.41 ng /mL and the limit of detectable concentrations is 0.01 ng/mL. We tested the 28 fluoroquinolones for cross reactivity and only ofloxacin (145%), marbofloxacin (82%), ofloxacin in its dextrorotatory form (68%), rufloxacin (67%), and garenoxacin (24%) had cross reactivity. The optimized ELISA technique allowed the detection of levofloxacin in milk from 0.33 ng/mL to 3.34 ng/mL. The recoveries were in the range of 89.5–102% with a relative standard deviation of 3%. We tested 45 real samples of milk purchased in local stores; for 5 of them the results were positive (near 1 ng/mL).     

Determination of telmisartan in human blood plasma: Part I: Immunoassay development

Telmisartan is an angiotensin II receptor antagonist and a known drug against high blood pressure. In this report, the development of a new and rapid analytical technique, an enzyme-linked immunosorbent assay (ELISA) for the determination of telmisartan in human blood plasma is described. The immunoassay is based on a conversion of 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) to 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA), which is detected by fluorescence spectroscopy. The limit of detection was 0.1 ng/mL, the limit of quantification was 0.3 ng/mL and the working range extended from 0.3 ng/mL to 300 ng/mL.     

Confirmatory analysis of acetylgestagens in plasma using liquid chromatography-tandem mass spectrometry

A confirmatory method has been developed and validated for the determination of chlormadinone acetate (CMA), megestrol acetate (MGA), melengestrol acetate (MLA) and medroxyprogesterone acetate (MPA) in bovine and porcine plasma. Analytes are extracted from plasma samples using matrix-assisted liquid-liquid extraction (LLE) on Extrelut NT columns followed by C18 solid-phase extraction (SPE). Analytes were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and quantification was performed using matrix-matched calibration standards in combination with deuterated internal standards. In accordance with Commission Decision 2002/657/EC, two ion transitions were monitored for each analyte. Decision limits (CCalpha) were estimated by analysing 20 blank plasma samples and ranged from 0.1 to 0.2 ng mL(-1). Detection capabilities (CCbeta) were estimated using 20 plasma samples fortified at 0.5 ng mL(-1) and were <0.5 ng mL(-1). In the range 0.5-2 ng mL(-1), the mean intra-laboratory reproducibility of the analytes ranged from 6 to 18% (%R.S.D.). Analytes were shown to be stable in fortified plasma samples for >8 months when stored at -20 degrees C.     

Bead injection ELISA for the determination of antibodies implicated in type 1 diabetes mellitus

This work introduces a novel analytical method for the detection and study of GAD65 autoantibodies, which have been implicated in the onset of type 1 diabetes. There is a clinical need for a rapid and automated assay for GAD65 autoantibodies. Therefore, this method was designed to exploit the advantages of bead injection (BI) analysis for enzyme-linked immunosorbent assays (ELISA). BI ELISA is a microscale technique that uses enzyme labeled secondary antibodies to detect the capture of target antibodies on immobilized antigen in the flow cell of the lab-on-valve (LOV) manifold. A detection limit of 20 ng mL(-1) of GAD65 monoclonal antibody 144 compares favorably with the sensitivity and precision of a standard ELISA currently employed to detect GAD65 autoantibodies. Compared to the standard ELISA protocol, BI ELISA offers a significantly reduced assay time and complete automation of solution handling and detection.     

Quantification of cephalexin as residue levels in bovine milk by high-performance liquid chromatography with on-line sample cleanup

A multidimensional, selective and precise high performance liquid chromatography (HPLC) method based on direct injection of biological samples has been developed for the determination of cephalexin in skimmed and whole bovine milk. The cephalosporin antibiotic was extracted from bovine milk using an octyl restricted access medium bovine serum albumin column (C₈-RAM-BSA) and analyzed on an octadecyl column using phosphate buffer (pH 7.5, 0.01 M): ACN (92:8, v/v) and ultraviolet detection at 260 nm. The calibration graph was linear in the concentration range of 25-1600 ng/mL and this is in accordance with the tolerance level of 100 ng/mL set by the FDA (Food and Drug Administration) and EU (European Union) for cephalexin as residue in bovine milk. The intra- and inter-day coefficients of variation for the replicate analysis at the quality control levels were less than 15% while the transfer efficiency was in the range of 90.2-92.3%. The limits of detection and quantification were 10 and 20 ng/mL, respectively.