Determination of olaquindox,carbadox and cyadox in animal feeds by ultra-performance liquid chromatography tandem mass spectrometry

Olaquindox, carbadox, and cyadox are chemically synthesised antibacterial and growth-promoting agents for animals. At high doses they may exert mutagenicity and hepatic and adrenal toxicities in animals. Regrettably, these substances are frequently abused or misused when added into animal feeds. Thus, developing a sensitive and reliable method for simultaneous determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds is crucially important for food safety monitoring. In this paper we optimised instrumental conditions, extraction solvents, solid phase extraction cartridges, and pH of the loading solvents on the Oasis HLB cartridge. Under the optimal conditions, mean recoveries ranged from 74.1 to 111%, and intra-day and inter-day variations were lower than 14.6% and 10.8%, respectively. The limits of quantification for olaquindox, carbadox, and cyadox were 0.05 mg kg^?1, 0.10 mg kg^?1, and 0.025 mg kg^?1, respectively. The proposed method uses ultra-performance liquid chromatography tandem mass spectrometry and is sensitive and reliable for the simultaneous determination of olaquindox, carbadox, and cyadox in three kinds of animal feeds (specifically, mixed feed, concentrated feed, and additive premixed feed). This method has good precision, high sensitivity, and good reproducibility, and thus it can be used for convenient and accurate determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds.     

Molecularly imprinted solid-phase extraction combined with high-performance liquid chromatography for analysis of trace olaquindox residues in chick feeds

BACKGROUND: Olaquindox, one of the antimicrobial growth accelerants, is usually used as a feed additive in livestock production to improve feed efficiency. Due to health concerns over possible carcinogenic, mutagenic and photoallergenic effects of olaquindox on animals, the development of a simple, rapid and sensitive analytical method for determination of olaquindox is crucial and necessary. RESULTS: In this paper, a novel and hydrophilic functionalised material of olaquindox‐imprinted polymer was synthesised in aqueous solution by a surface molecular imprinting in combination with a sol–gel process. This imprinted material was characterised by Fourier transform infrared, scanning electron microscopy, and static and kinetic adsorption experiments, and results showed that it had good recognition and selective ability, and fast adsorption‐desorption dynamics for olaquindox. Applying the prepared material as sorbent, a method of molecularly imprinted solid‐phase extraction (MISPE) for separation and analysis of olaquindox residues in feeds coupled with HPLC was presented. Under the selected MISPE condition, the detection limit (S/N = 3) for olaquindox was 68.0 ng L^?1, the RSD for five replicate extractions of 50 µg L^?1 olaquindox was 9.8%. The blank chick feed samples spiked with olaquindox at 0.0025 and 0.010 mg g^?1 levels were extracted and determined by the developed method, with recoveries ranging from 90% to 96%. CONCLUSION: This method was applied for enrichment and analysis of olaquindox in animal feed samples with good accuracy and repeatability. This study will provide a sensitive and fast method for the monitoring of olaquindox residues in foods. Copyright © 2011 Society of Chemical Industry     

A method for the detection and estimation of trace levels of theobromine in mixed horse feed

A method for the detection and estimation of theobromine in compounded horse feed by high-performance liquid chromatography (h.p.l.c.) is presented. The sample is extracted by refluxing with distilled water and clarified by the addition of Carrez reagents. The level of co-extractives is reduced by precipitation by the addition of 4 vols of acetone, and a measured volume of the filtrate transferred on to a silica column for clean-up. The theobromine is eluted with chloroform (85%)-propan-2-ol (15%) and the eluate evaporated to dryness. The residue is dissolved in water and analysed by h.p.l.c. (using a reverse-phase column and ultraviolet detection at 272 nm). The method has been tested on mixed horse feeds and their ingredients. Theobromine recoveries were in the range 78–86% and concentrations down to 2 mg kg^?1 could be measured. Three ways to confirm positive results are presented.     

Label-free gold nanoclusters as quenchable fluorescent probes for sensing olaquindox assisted by glucose oxidase-triggered Fenton reaction

Glucose oxidase (GOx) catalyses oxidation of glucose accompanied with the generation of hydrogen peroxide. With the addition of Fe²⁺, hydroxyl radical produced by Fenton reaction between hydrogen peroxide and Fe²⁺ may quench the fluorescence of gold nanoclusters. In this work, a fluorescent enzyme-linked immunosorbent assay with gold nanoclusters was designed with a straightforward signal output, in which the fluorescence of gold nanoclusters was quenched by GOx-triggered Fenton reaction. Olaquindox was selected as a target analyte. Gold nanoclusters capped with bovine serum albumin and GOx-linked olaquindox conjugates were successfully prepared. Olaquindox in samples directly competed with the GOx-linked olaquindox conjugates for binding immobilized antibody. Consequently, the fluorescence signal increased with the amount of olaquindox. Under optimal conditions, the fluorescent enzyme-linked immunosorbent assay exhibited a favorable performance to detect olaquindox in swine feeds, demonstrating a good linear range from 1.0 µg kg^?1 to 150 µg kg^?1 with a reliable correlation coefficient (R² = 0.9918); the limit of detection was 0.68 µg kg^?1. Average recoveries in spiked samples were 85.3% to 113.5%. The proposed strategy is a promising approach for the detection of olaquindox and other harmful small molecules.     

Quantitative analysis of styrene monomer in foods. A limited East Australian Survey

The levels of styrene monomer in foods packaged in polystyrene containers were determined by a headspace gas chromatography (g.c.) method and two reversed phase high-performance liquid chromatography (h.p.l.c.) methods. A total of 146 samples were analysed from Victoria and New South Wales which included yoghurt, cream, cheese, dessert, ice cream, egg white, onion dip and margarine. The highest level of styrene found was 0.1 mg kg^?1 in yoghurt. About 85% of all yoghurt samples were found to have values less than 0.05 mg kg^?1. The lowest values of styrene obtained were for margarine samples, of which more than 90% contained less than 0.010 mg kg^?1. The estimated limits of detection of the h.p.l.c. methods for all products except margarine were 0.005 mg kg^?1. The h.p.l.c. detection limit for margarine and the g.c. method for yoghurt were estimated to be 0.01 mg kg^?1.     

Development of an on-line molecularly imprinted chemiluminescence sensor for determination of trace olaquindox in chick feeds

BACKGROUND: Olaquindox, as one of the antimicrobial growth accelerants, is usually used in livestock production to improve feed efficiency. Due to health concerns over possible carcinogenic, mutagenic and photoallergenic effects of olaquindox on animals, the development of simple, rapid and sensitive analytical method for determination of olaquindox is crucial and necessary. RESULTS: In this study, a surface molecularly imprinted polymer was prepared by a molecular imprinting technique in combination with a sol‐gel process using activated silica gel as a support material. This imprinted material exhibited with good recognition and selective ability, and fast adsorption‐desorption dynamics toward olaquindox. Using it as the recognition element, a new on‐line molecularly imprinted solid phase extraction coupled with chemiluminescence sensor for the determination of olaquindox was developed. The factors affecting preconcentration of the analytes and sensitivity of the method were all investigated. Under the optimal condition, the linear range of the calibration graph was between 2 × 10^?8 and 1 × 10^?6 g mL^?1, and the detection limit of this method was 7 × 10^?9 g mL^?1. The blank chick feed samples spiked with olaquindox at 0.3, 0.9 and 1.5 µg g^?1 levels were extracted and determined by this presented method with recoveries ranging from 87% to 94%. This method was validated by high‐performance liquid chromatography and the results correlated well with those obtained by both methods. Moreover, this method was quantitatively analysed with two contaminated chick feed samples. CONCLUSION: This study will provide a sensitive and fast method for the monitoring of olaquindox residues in foods. Copyright © 2012 Society of Chemical Industry     

Preparation and Application of a Molecular Imprinting Matrix Solid Phase Dispersion Extraction for the Determination of Olaquindox in Chicken by High Performance Liquid Chromatography

In this paper, a new method was established to extract olaquindox in chicken by matrix solid phase dispersion extraction using the molecularly imprinted polymers as solid phase materials. The polymers were prepared using olaquindox as the template, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linking agent. The imprinted material was characterized by static and kinetic adsorption experiments. The results showed that it had good recognition, selective ability, and fast adsorption–desorption dynamics for olaquindox. The prepared material was used as solid phase materials of matrix solid phase dispersion to enrich the olaquindox and then determined by high performance liquid chromatography. Under the optimized conditions, the range of recovery spiked with olaquindox at 1.0 and 2.0 μg?g^?1 levels was between 85.3 and 93.2 %.     

Use of high performance liquid chromatography combined with fluoresence detection for the identification and estimation of aflatoxins and ochratoxin in food

A method has been developed for the separation, identification and estimation of six mycotoxins in foods at the μg kg^?1 level. The food is extracted by using aqueous acetonitrile and the extract is then washed with 2,2,4-trimethylpentane and any mycotoxins re-extracted into chloroform. The extract is then purified using grooved t.l.c. plates coated with silica gel. After chromatography, any fluorescent bands are removed and examined by h.p.l.c. using a fluorescence detector. The method has been tested using a range of foods including total diet samples. Recoveries at twice the detection limit varied from 40 to 95%.     

H.p.l.c. Determination of trace levels of succinic acid in orange juice from Freeze-damaged and undamaged fruit

Succinic acid was determined by high-performance liquid chromatography (h.p.l.c.) in juice from oranges subjected to freeze damage on the tree and by storage at below freezing temperatures. Succinic acid levels did not change in fruit subjected to freezing temperatures while on the tree or in storage. In order to measure succinic acid at these low levels (5–180 mg litre^?1 juice) an earlier h.p.l.c. procedure was modified.     

Determination of fructose and glucose in cucurbitaceae by h.p.l.c.

High performance liquid chromatography (h.p.l.c.) was used to characterise and quantify fructose and glucose in seven members of the Cucurbitaceae family. The h.p.l.c. system consisted of a μ-Bondapak/Carbohydrate packed column, a solvent system of acetonitrile-water (800 ml l^?-1) with a flow rate of 2.5 ml min^?-1, and a refractive index detector. The analysis, including sample preparation, was completed in less than 45 min. The quantities of fructose and glucose obtained were, except for one sample, <1% on a fresh weight basis, and the glucose: fructose ratios ranged from 0.41:1 to 0.93:1.     

The high performance liquid chromatographic determination of total malondialdehyde in vegetable oil with dansyl hydrazine

A simple high-performance liquid chromatographic (h.p.l.c.) method has been developed for determining total malondialdehyde (MDA), including free MDA and MDA released from its precursor in vegetable oils. Free MDA was reacted with dansyl hydrazine in an acidic medium, and the product, 1-dansyl-pyrazole(DP), was determined by h.p.l.c. The reaction releasing MDA from its precursor requires Fe³⁺ in 5% hydrochloric acid medium. Recoveries of free MDA and tetramethoxypropane (used as an MDA precursor) in vegetable oils were satisfactory. During autoxidation, a remarkable difference between the DP values of methyl oleate, methyl linoleate and some vegetable oils, and that of methyl linolenate was observed. Only methyl linolenate released MDA during autoxidation. For autoxidised methyl linolenate the DP value correlated with the thiobarbituric acid(TBA) value, although the DP value was only 30% of the TBA value.     

The assay of the coccidiostat clopidol in animal feeds by high-pressure liquid chromatography

A simple method for the assay of the coccidiostat clopidol by high performance liquid chromatography (h.p.l.c.) is described. After extraction the solution is neutralised and filtered and injected on the h.p.l.c.-column. It is quantified by u.v.-detection. The method has been tested on commercial feed samples. The agreement with a standard reference method is good. The precision of the proposed method has a standard deviation of 2%. The recovery of added clopidol is 101% with a standard deviation of 2.4%. No interferences to the method have been found.     

Separation of fructose,glucose and sucrose in fruit by high performance liquid chromatography using UV detection at 190 nm

Fructose, glucose and sucrose were separated by high performance liquid chromatography (h.p.l.c.) on a 5 μm amine column and detected by absorbance at 190 nm. The procedure quantified these sugars in orange juice and in carambola fruit, but preliminary ion exchange column chromatography was necessary before satisfactory h.p.l.c. results could be obtained. The method is sensitive to 1.2 μg of fructose or 9 μg of glucose or sucrose.     

Assessment of a procedure for fractionating organic phosphates in soil and organic materials using gel filtration and h.p.l.c

A method for extracting and fractionating organic phosphates is described. In the fractions, inositol hexaphosphate (IHP) was determined with a carbon detector after high pressure liquid chromatography (h.p.l.c). Sample preparation consisted of a single mild extraction with 0.1M EDTA in 0.3M-NaOH and a preseparation by gel filtration. Adenosine-5-triphosphate (ATP) could also be separated and determined in this way. With a number of samples, clean-up by gel filtration could not prevent deterioration of the anion exchange column, used in the h.p.l.c. determination. IHP was then determined as total P after isolation by two consecutive gel filtrations. Limits of detection were 5-50 μg P/g when determining IHP by h.p.l.c. and 0.05-0.5 μg P/g when determining IHP as total P after isolation by gel filtration. Limits of detection when determining ATP by h.p.l.c. were 5-50 ng P/g. Results of applying the method to soil and various organic materials are given and discussed.     

Simultaneous determination of five quinoxaline-1,4-dioxides in animal feeds using an immunochromatographic strip

An immunochromatographic (ICG) strip was developed for the simultaneous quantitative determination of five quinoxaline-1,4-dioxides in animal feed. For this purpose, polyclonal antibodies (PcAb) with group-specific quinoxaline-1,4-dioxides were conjugated to colloidal gold particles as the detection reagent for ICG strips to test for quinoxaline-1,4-dioxides. This method achieved semi-quantitative detection of quinoxaline-1,4-dioxides within 5–10 min. The visual lower detection limits of the strip for quinocetone, cyadox, carbadox, mequindox and olaquindox were 10, 15, 15, 20 and 20 ng ml^?1, respectively. Using an ICG strip reader, the 50% inhibitions (IC₅₀ values) were calculated to be 9.1, 13.5, 16.6, 20.2 and 21.3 ng ml^?1 for quinocetone, cyadox, carbadox, mequindox and olaquindox, respectively. When used to analyse samples of animal feed, acceptable recovery rates of 77.5–99.5% and coefficients of variation (CVs) of 4.3–10.7% were obtained. Levels measured with the ICG strip for 10 spiked samples were confirmed by HPLC with a high correlation coefficient of 0.9965 (n = 10). In conclusion, the method was rapid and accurate for simultaneous determination of five quinoxaline-1,4-dioxides antibiotics in animal feed.     

Determination of sugars in tropical root crops using 13C N.m.r. spectroscopy: Comparison with the h.p.l.c. method

The ¹³C nuclear magnetic resonance (n.m.r.) spectrum of sugars normally present in foods, viz. glucose, fructose, sucrose, maltose and raffinose contains resonances that are diagnostic of each sugar. The quantity of each sugar is determined by measurement of the ratios of the peak heights of the sugar resonances compared with peak height of an internal standard, present at constant concentration in all solutions. A linear calibration relationship between the peak height ratio and concentration is established for single resonances of glucose and fructose and for two resonances of each of sucrose, maltose and raffinose. Samples of sweet potato and taro flour dried at 40°C, were blended with 80% ethanol, the sugars were extracted and analysed by ¹³C n.m.r. and h.p.l.c. Both methods gave similar results and have similar degrees of reproducibility. The n.m.r. method is useful for quantitation of sugars and identification of other molecules extracted from the food, but requires more material than does h.p.l.c. Drying at 40°C causes a decrease in sucrose content and increases in glucose, fructose and maltose which partly compensate for the loss in sucrose. There is considerable variability of the sugar content between different sweet potato tubers and between different taro corms of the same cultivar as well as across different cultivars. For sweet potato the total sugar content is 0.6-3.6% fresh weight, with considerably more sucrose present than glucose, fructose and maltose. With taro total sugar content is 0.2-0.8% fresh weight. Raffinose is present in one sample only.     

In vivo studies to highlight possible illegal treatments of rabbits with carbadox and olaquindox

For the treatment of rabbit dysentery and bacterial enteritis, veterinary practitioners often adopt veterinary medicinal products authorised for other food-producing species, but in some cases non-authorised drugs frequently used in the past, such as carbadox and olaquindox, might be illegally adopted. To verify the carbadox and olaquindox distribution and persistence in rabbit tissues, two independent in vivo studies were carried out. In the first study, 24 healthy rabbits received water medicated with carbadox at 100 mg l^?1 over a period 28 days, whereas in the second one, 24 healthy rabbits were administered water containing olaquindox at 100 mg l^?1. In each study rabbits were randomly assigned to four groups to be sacrificed respectively at 0, 5, 10 and 20 days from treatment withdrawal, for depletion studies. A control group of six animals was adopted for control and as a reservoir of blank tissues. Muscle and liver samples collected from each treated animal were stored at ?20°C pending the analysis. Sensitive and robust liquid chromatography-tandem mass spectrometry analytical methods were set up for the parent compounds and their main metabolites quinoxaline-2-carboxylic acid, desoxycarbadox and 3-methylquinoxaline-2-carboxylic acid to verify their residual. Data collected demonstrate that the combination of liver as target matrix, quinoxaline-2-carboxylic acid and 3-methylquinoxaline-2-carboxylic acid as marker residue and enzymatic digestion is strategic to evidence carbadox and/or olaquindox illegal treatments in rabbits, even 20 days after treatment withdrawal at concentration levels higher than 0.5 µg kg^?1. This findings suggests that liver should be proposed as target matrix for official control in national monitoring plan.     

Use of high performance liquid chromatography for the identification and estimation of zearalenone, patulin and penicillic acid in food

A method is described for the estimation of 3 u.v.-absorbent mycotoxins in foods by using semi-preparative and analytical h.p.l.c. columns. The foodstuff is extracted using aqueous acetonitrile and fatty matter is removed from the extract by partition into 2,2,4-trimethylpentane. Mycotoxins present in the fat-free extract are then back extracted into chloroform and separated into two fractions on a silica gel column using (a) diethyl ether and (b) chloroform: methanol. Further purification on a semi-preparative alumina column is described prior to measurement of mycotoxin content on analytical h.p.l.c. columns. Recoveries of zearalenone, patulin and penicillic acid added at 2 to 4 times the detection limit to samples of meat, dairy products and cereals ranged from 50 to 100%.     

Validation of an analytical method for the determination of carbadox and olaquindox in feedstuff by liquid chromatography coupled to UV and/or diode array detection

The performance characteristics of an analytical method based on high-performance liquid chromatography (HPLC) for the detection of the banned growth promoters, carbadox and olaquindox, in feedstuff were determined via a collaborative study. The relative standard deviation of repeatability (RSD_r) ranged 1.1-5.5% for carbadox and 2.5-6.2% for olaquindox. The relative standard deviation of reproducibility (RSD_R) ranged 6.4-10.7% for carbadox and 12.8-20.0% for olaquindox. In all cases, the HORRAT values were equal or below the critical value of 1.5. Moreover, trueness in all cases was between the acceptance limits of 80 and 110%. Consequently, it was concluded that the method is suitable for quantitative evaluation. The method was also qualitatively assessed in terms of correct identification of the target analytes by examination of the UV spectrum when the more specific diode array detector was coupled to HPLC. In all cases, the percentage of correct identifications was ≥94% for olaquindox and carbadox, while the percentage of false negatives was ≤6%, suggesting the extended utilization of the HPLC method from quantitative to confirmatory status with a diode array detector.     

Simultaneous determination of vitamins A and E in feeds and foods by reversed phase high-pressure liquid chromatography

A simple high-pressure liquid chromatographic method for the simultaneous determination of vitamins A and E in animal feeds and foods has been developed and evaluated. After saponification and extraction the sample was run on a reversed-phase column with water-methanol as the mobile phase. The detector was an u.v.-lamp at 280 nm. The method has been tested on different types of feeds and foods containing between 2-30 μg vitamin A g^?1 of sample and 5-300 μg vitamin E g^?1 of sample. The recovery of added vitamins was 86 % for retinylacetate and 91 % for α-tocopheryl-acetate with a standard deviation of 3 % for both. Results of the proposed procedure agreed well with those obtained by other methods.