Time-resolved luminescence screening of antibiotics in tissue matrices without centrifugation and filtration: spiked recovery studies

Analyses of chemical residues in animal tissue matrices require multistep sample preparation. To simplify this process, a methodology was developed that combines sorbent extraction and solid-matrix time-resolved luminescence (TRL); it was applied to tetracycline screening in milk. Reported here is an effort to extend its application to tissue matrices, illustrated by oxytetracycline (OTC) screening in catfish muscle. Extraction and enrichment are accomplished by immersing small C18 sorbent strips into tissue homogenates for 20 min, followed by a 3 min rinse in water and a 2 min dip in a reagent solution. After desiccation, TRL is measured directly on the sorbent surface. Tissue particulates no longer interfere via attenuation or scattering, rendering centrifugation and filtration unnecessary. The integrated TRL intensity shows a linear dependence on OTC concentration in the 0-8 microg/g range (R2 = 0.9992) with a 0.026 microg/g limit of detection. To screen OTC at 2 microg/g, the U.S. regulatory tolerance level, a threshold is established at x2-3sigma2, where x2 and sigma2 are the mean and standard deviation, respectively, of the TRL signals from 15 samples fortified at 2 microg/g. Among 45 blind samples randomly fortified at 0-4 microg/g, 41 were screened correctly and 4 negative samples were presumed positive. This method has the potential to improve throughput and save assay costs by eliminating acids, organic solvents, centrifugation, and filtration.     

Hyphenation of sorbent extraction and solid-matrix time-resolved luminescence using tetracycline in milk as a model analyte

Hyphenation of sorbent extraction and solid-matrix time-resolved luminescence (TRL) was demonstrated using tetracycline (TC) in milk as a model analyte. The performance of a C18-impregnated silica layer was evaluated as both an extraction sorbent and a TRL substrate. To extract TC, a 10 x 6 mm glass-backed C18 layer was dipped into a 10 mL milk sample for 10 min followed by a 3-min water immersion for cleanup. The sorbent was then spotted with a TRL reagent solution at pH 9 that contained 5 mM europium nitrate and 5 mM EDTA. After a brief desiccation period, TRL was measured directly on the sorbent surface with a commercial fluorescence spectrophotometer. By eliminating the need to elute the analyte from the sorbent, organic solvent was not needed and sample preparation was greatly simplified. The integrated signal showed a linear dependence (R(2) = 0.9938) on TC concentration in the 0-3000 microg/L range. The same protocol was applicable to screening TC in fat-free, 2% low-fat, and whole milk at 300 microg/L, the US. regulatory tolerance level set by the Food and Drug Administration (FDA). This easy, fast, and low-cost screening method is friendly to the environment and particularly suitable for liquid samples.     

Liquid chromatographic determination of alpha-zearalenol and zearalenone in corn: collaborative study

The liquid chromatographic determination of alpha-zearalenol and zearalenone in corn was collaboratively studied. Each of 13 collaborators received 7 corn samples; 2 were blanks and 5 were spiked to contain 50, 100, and 200 ng alpha-zearalenol/g and 50, 100, 500, 1000, and 4000 ng zearalenone/g. Four sets (including blanks) of blind duplicates were included in the study. Five naturally contaminated corn samples (one in duplicate) were also provided. All collaborators detected both mycotoxins at 50 ng/g. Average recoveries reported by all collaborators ranged from 81.9% at 200 ng/g to 100.3% at 50 ng/g for alpha-zearalenol and from 77.8% at 1000 ng/g to 123% at 50 ng/g for zearalenone. Three collaborators reported false positives for both alpha-zearalenol and zearalenone. The within-laboratory CV values based on blind duplicates were 22.6% for alpha-zearalenol and 31.4% for zearalenone. The CV values based on laboratory-sample interaction were 25.6 and 33.8% for alpha-zearalenol and zearalenone, respectively. The CV values for naturally contaminated samples (including duplicates) were 47.0% for alpha-zearalenol and 37.7% for zearalenone. The method has been adopted official first action.     

Co-occurrence of ochratoxin A and citrinin in cereals from Bulgarian villages with a history of Balkan endemic nephropathy

Cereal samples were collected in 1998 from Bulgarian villages without [control village (C), n = 20] or with [endemic villages (E); E1, n = 21; E2, n = 30; E3, n = 23] a history of Balkan endemic nephropathy (BEN). Sampling included foods (wheat, corn) and feeds (barley, oats, wheat bran). Analysis of ochratoxin A and citrinin was done by enzyme immunoassays (EIA), with detection limits of 0.5 and 5 ng/g, respectively. Ochratoxin A-positive results were confirmed by HPLC after immunoaffinity chromatography. Highest toxin levels were found in wheat, wheat bran, and oats. For ochratoxin A, the percentages of positives were 35% (C), 29% (E1), 30% (E2), and 47% (E3), the mean/median values of positives were 1.5/1.3 ng/g (C), 11/1.6 ng/g (E1), 18/1.6 ng/g (E2), and 3.5/1.5 ng/g (E3). For citrinin, 5.0% (C), 14% (E1), 3.3% (E2), and 13% (E3) were positive, and the mean/median values were 6.1/6.1 ng/g (C), 180/83 ng/g (E1), 10/10 ng/g (E2), and 84/20 ng/g (E3). Highest concentrations of ochratoxin (maximum = 140 ng/g) and citrinin (maximum = 420 ng/g) were found in samples from endemic villages. Co-contamination with ochratoxin A and citrinin was found for one sample (14% of positives) from village C and for six samples (22% of positives) from villages E1-E3. Citrinin levels in these samples were 2-200 times higher than those of ochratoxin A.     

Ultrasensitive detection of genetically modified maize DNA by capillary gel electrophoresis with laser-induced fluorescence using different fluorescent intercalating dyes

In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.     

Monoclonal enzyme immunoassay for the analysis of carbaryl in fruits and vegetables without sample cleanup

The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput.     

Simultaneous quantitation of 2-acetyl-4-tetrahydroxybutylimidazole, 2- and 4-methylimidazoles, and 5-hydroxymethylfurfural in beverages by ultrahigh-performance liquid chromatography-tandem mass spectrometry

An ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometric (MS/MS) method was developed for the simultaneous quantification of 2-acetyl-4-tetrahydroxybutylimidazole (THI), 2- and 4-methylimidazoles (2-MI and 4-MI), and 5-hydroxymethylfurfural (HMF) in beverage samples. A C30 reversed-phase column was used in this method, providing sufficient retention and total resolution for all targeted analytes, with an MS/MS instrument operated in selected reaction monitoring (SRM) mode for sensitive and selective detection using isotope-labeled 4-methyl-d(3)-imidazole (4-MI-d(3)) as the internal standard (IS). This method demonstrates lower limit of quantification (LLOQ) at 1 ng/mL and coefficient of determination (r(2)) >0.999 for each analyte with a calibration range established from 1 to 500 ng/mL. This method also demonstrates excellent quantification accuracy (84.6-105% at 5 ng/mL, n = 7), precision (RSD < 7% at 5 ng/mL, n = 7), and recovery (88.8-99.5% at 10, 100, and 200 ng/mL, n = 3). Seventeen carbonated beverage samples were tested (n = 2) in this study including 13 dark-colored beverage samples with different flavors and varieties and 4 light-colored beverage samples. Three target analytes were quantified in these samples with concentrations in the range from 284 to 644 ng/mL for 4-MI and from 706 to 4940 ng/mL for HMF. THI was detected in only one sample at 6.35 ng/mL.     

Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables

The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput.     

Ultratrace analysis of nine macrolides, including tulathromycin A (Draxxin), in edible animal tissues with minicolumn liquid chromatography tandem mass spectrometry

The analysis of nine macrolides is presented, including tulathromycin A (Draxxin), in beef, poultry, and pork muscle with a simple multiresidue extraction and analysis method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The sample preparation method involves extraction with acetonitrile and defatting with hexane followed by dilution of the extracts for analysis. Separation of the nine macrolides was performed using an Atlantis dC 18, 3 mum, 3.9 mm x 20 mm minicolumn (guard column). Detection was carried out with two multiple reaction monitoring experiments per macrolide. The method detection limits (MDLs) were based on three times standard deviation of eight repeat spikes at 3.0 ng/g of a mix of the nine macrolides in the various tissues. The MDLs and retention times for the macrolides were as follows: lincomycin, 0.19 ng/g (t R = 5.00 min); tulathromycin, 0.46 ng/g (t R = 5.63 min); spiramycin, 0.21 ng/g (t R = 6.06 min); pirlimycin, 0.10 ng/g (t R = 6.04 min); clindamycin, 0.16 ng/g (t R = 6.20 min); tilmicosin, 0.29 ng/g (t R = 6.38 min); erythromycin, 0.19 ng/g (t R = 6.62 min); tylosin, 0.10 ng/g (t R = 6.72 min); and josamycin, 0.09 ng/g (t R = 6.98 min). Precision at 25 ng/g (n = 4) ranged from 2.3 to 9.4% for the compounds from beef muscle. Of interest is the detection of incurred residues of tulathromycin A in edible calf tissue at 0.10-7 mug/g, which is presented here for the first time.     

Rapid screening method for deoxynivalenol in agricultural commodities by fluorescent minicolumn

A rapid screening procedure based on the selective adsorption of deoxynivalenol (DON) from extracts of wheat and corn has been developed. DON is extracted from the sample with acetonitrile-water (85 + 15) and partially purified on a preparative minicolumn. Solvent is evaporated and the residue is dissolved in toluene-acetone (95 + 5) and chromatographed on a novel detector minicolumn which selectively adsorbs DON. A blue fluorescence is produced when the column is heated 5 min at 100 degrees C. The procedure is capable of detecting DON at greater than or equal to 500 ng/g. Forty-three wheat samples, contaminated with DON at 60-6300 ng/g, were assayed by gas chromatography-mass spectroscopy (GC-MS) of the heptafluorobutyryl derivative of DON and by the selective adsorption procedure. Comparison of results showed 91% agreement between data from the 2 methods. Selective adsorption assays were positive for all samples that were greater than or equal to 500 ng/g by GC-MS (no false negatives) and were negative for 85% of samples less than 500 ng/g (4/27 false positives). These four samples contained greater than 200 ng/g by GC-MS. Samples of wheat (64), corn (23), soybeans (8), and sorghum (6) were extracted and extracts were assayed by thin-layer chromatography and the selective adsorption procedure. Selective adsorption assays agreed with TLC results.     

Synthesis of a novel composite imprinted material based on multiwalled carbon nanotubes as a selective melamine absorbent

A novel composite imprinted material, on the basis of a multiwalled carbon nanotube (CNT)-incorporated layer using melamine as a template, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linker, was synthesized by a surface imprinting technique. The imprinted/CNT sorbent was characterized by a scanning electron microscope (SEM). Adsorption dynamics and a Scatchard adsorption model were employed to evaluate the adsorption process. The results showed that the imprinted/CNT sorbent displayed a rapid dynamic adsorption and a high adsorption capacity of 79.9 μmol g(-1) toward melamine. Applied as a sorbent, the imprinted/CNT sorbent was used for the determination of melamine in a real sample by online solid-phase extraction-high-performance liquid chromatography (SPE-HPLC). An enrichment ratio of 563-fold, detection limit (S/N = 3) of 0.3 μg L(-1), and quantification limit of 4.5 μg L(-1) were achieved.     

Liquid chromatographic determination of olaquindox in medicated feeds and in contents of porcine gastrointestinal tract

A liquid chromatographic method for the determination of olaquindox in both medicated feeds and porcine gastrointestinal tract is described. Samples are extracted with water and cleaned on a disposable reverse-phase column. The eluate is chromatographed on a reverse-phase column under isocratic conditions. Olaquindox is detected by UV absorption at 260 nm. The minimum amount detected by this method was 0.075 ng. The corresponding minimum detectable concentration in a 1 g sample was 0.3 mg/kg. The detector response was linear within the interval of 0-500 ng. Mean recovery of olaquindox in spiked gastrointestinal samples was 89 +/- 5% (mean +/- standard deviation, n = 43). Concentration profiles of olaquindox in the gastrointestinal tract of pigs fed medicated feed were used to evaluate the preventive potency against Treponema hyodysenteriae. The presence of some N-O reduced metabolites of olaquindox in the gastrointestinal tract was assessed.     

Chloramphenicol extraction from honey, milk, and eggs using polymer monolith microextraction followed by liquid chromatography-mass spectrometry determination

A rapid confirmatory method for monitoring chloramphenicol (CAP) residues in honey, whole milk, and eggs is presented. This method is based on the polymer monolith microextraction (PMME) technique and high-performance liquid chromatography (HPLC)-electrospray ionization mass spectrometry (MS). A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium. To obtain optimum extraction efficiency, several parameters related to PMME were investigated. After dissolution in 20 mM phosphate solution at pH 4.0 and centrifugation, honey, eggs, or milk samples were directly passed through the extraction tube. The LC-MS instrument was equipped with an electrospray ion source and a single quadrupole. The eluates were analyzed by LC-MS in the negative-ion mode and by monitoring a pair of isotopic ions for the target compound. The in-source collision-induced dissociation process produced confirmatory ions. The recoveries of CAP from real samples spiked at 0.1-10 ng/g (honey), 0.2-10 ng/mL (milk), and 0.2-10 ng/g (egg) were in the range of 85-102%, with relative standard deviations ranging between 2.1% and 8.9%. The limits of detection (S/N = 3) were 0.02 ng/g, 0.04 ng/mL, and 0.04 ng/g in honey, milk, and eggs, respectively. The proposed method was proved to be robust in monitoring CAP residue in honey, milk, and eggs.     

Liquid chromatographic method for determination of citreoviridin in corn and rice

Citreoviridin, a neurotoxic mycotoxin, has been found as a natural contaminant in corn left unharvested in the southeastern United States and in rice of several Asian countries, including Japan. A reliable analytical method for the quantitative determination of citreoviridin in corn and rice is described. Corn or rice is extracted with dichloromethane, and the extract is partially purified on silica and amino solid-phase extraction (SPE) columns. The extract is analyzed for citreoviridin by normal-phase liquid chromatography, using a mobile phase of ethyl acetate-hexane (75 + 25) at 1.5 mL/min and a fluorescence detector to measure the yellow fluorescence (388 nm excitation, 480 nm emission). With a 100 microL injection loop, the relationship between concentration and injection volume is linear for 20-60 microL injections. Recoveries of citreoviridin added to yellow corn at 10-50 ng/g were 91.0-96.9%; recoveries from white corn (10-50 ng/g added) were 96.8-102.8%. Recoveries of 5000 ng/g added to white corn were 89.0%, indicating that heavily contaminated samples can be assayed by the method. Minimum detection limits were 10 ng for citreoviridin standard and 2 ng/g for citreoviridin added to corn. White rice fermented with Penicillium citreo-viride (1524 ppm) was mixed with and serially diluted with uncontaminated ground corn to obtain citreoviridin-contaminated corn (ca 25 ppb). When the samples were assayed by the method, a mean level of 24.4 +/- 1.65 ppb (6.5% coefficient of variation) was obtained. Four fermented rice food samples and 3 commercial rice samples were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of moniliformin in maize plants using hydrophilic interaction chromatography

A novel HPLC method was developed for detection of the Fusarium mycotoxin, moniliformin in whole maize plants. The method is based on hydrophilic interaction chromatography (HILIC) on a ZIC zwitterion column combined with diode array detection and negative electrospray mass spectrometry (ESI(-)-MS). Samples were extracted using acetonitrile-water (85:15), and the extracts were cleaned up on strong anion exchange columns. By this procedure we obtained a recovery rate of 57-74% moniliformin with a limit of detection at 48 ng/g and a limit of quantification at 96 ng/g using UV detection at 229 nm, which is comparable to current methods used. Limit of detection and quantification using ESI(-)-MS detection was 1 and 12 ng/g, respectively. Screening of maize samples infected with the moniliformin producing fungi F. avenaceum, F. tricinctum, or F. subglutinans detected moniliformin levels of 1-12 ng/g in 15 of 28 samples using ESI(-)-MS detection. To our knowledge this is the first example of HILIC separation in mycotoxin analysis.     

Rapid determination of estrogens in milk samples based on magnetite nanoparticles/polypyrrole magnetic solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

In this study, a nanocomposite of polypyrrole-coated magnetite nanoparticles (denoted as MNPs/PPy) was prepared and employed as magnetic solid-phase extraction (MSPE) sorbent for extraction of estrogens from milk samples. Because the polypyrrole coating possessed a highly π-conjugated structure and hydrophobicity, MNPs/PPy showed excellent performance for the estrogen extraction. Estrogens could be captured directly by MNPs/PPy from milk samples without protein precipitation. Moreover, the extraction could be carried out within 3 min. Thus, a rapid, simple, and effective method for the analysis of estrogens in milk samples was established by coupling MNPs/PPy-based MSPE with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limits of detections for estrogens investigated were in the range of 5.1-66.7 ng/L. The recoveries of estrogens (concentration range of 0.5-20 ng/mL) from milk samples were in the range of 83.4-108.5%, with relative standard deviations ranging between 4.2 and 15.4%.     

Rapid method for quantitative analysis of the aroma impact compound, 2-acetyl-1-pyrroline, in fragrant rice using automated headspace gas chromatography

A rapid method employing static headspace gas chromatography (HS-GC) has been developed and validated for quantitative analysis of the impact aroma compound, 2-acetyl-1-pyrroline (2AP), in grains of fragrant rice. This developed method excludes wet extraction, and the rice headspace volatiles are brought directly and automatically to GC analysis. The conditions of the static HS autosampler were optimized to achieve high recovery and sensitivity. The most effective amount of rice sample used was 1 g, which provided 51% recovery and a linear multiple headspace extraction (MHE) plot of the peak area of 2AP. The sensitivity of the method was enhanced by utilizing a megabore fused silica capillary column in conjunction with a nitrogen-phosphorus detector (NPD). Method validations performed for both static HS-GC-FID and HS-GC-NPD demonstrated linear calibration ranges of 20-10 000 (r(2) = 0.9997) and 5-8000 (r(2) = 0.9998) ng of 2AP/g of rice sample, respectively. The limits of detection for both systems were 20 and 5 ng of 2AP, and the limits of quantitation were 0.30 and 0.01 g of brown rice sample, respectively. Reproducibility calculated as intraday and interday coefficients of variation were 3.25% RSD (n = 15) and 3.92% RSD (n = 35), respectively, for SHS-GC-FID and 1.87% RSD (n = 15) and 2.85% RSD (n = 35), respectively, for SHS-GC-NPD. The method was found to be effective when applied to the evaluation of aroma quality, based on 2AP concentrations, of some fragrant rice samples.     

Improved high-performance liquid chromatography (HPLC) method for qualitative and quantitative analysis of allantoin in Zea mays

A high-performance liquid chromatography (HPLC) method for the qualitative and quantitative analysis of allantoin in silk and seed of Zea mays has been developed. Allantoin separation in crude extract was achieved using a C 18 column and phosphate buffer solution (pH 3.0) as a mobile phase at ambient temperature at a flow rate of 1.0 mL/min and detected at 210 nm. The results showed that the amount of allantoin in samples was between 14 and 271 mg/100 g of dry plant material. A comprehensive validation of the method including sensitivity, linearity, repeatability, and recovery was conducted. The calibration curve was linear over the range of 0.2-200 microg/mL with a correlation coefficient of r2>0.999. Limit of detection (LOD, S/N=3) and limit of quantification (LOQ) values of the allantoin were 0.05 and 0.2 microg/mL (1.0 and 4.0 ng) respectively. The relative standard deviation (RSD) value of the repeatability was reported within 1.2%. The average recovery of allantoin added to samples was 100.6% with RSD of 1.5%.     

Determination of fluoroquinolones in milk samples by postcolumn derivatization liquid chromatography with luminescence detection

A liquid chromatography (LC) method with luminescence detection for the determination of eight quinolone antibiotics is reported. The system encompasses three consecutive steps: (a) chromatographic separation using reverse-phase mode (RP-LC), (b) postcolumn derivatization reaction, and (c) luminescence detection by monitoring fluorescence (FL) and time-resolved (TR) signals. The derivatization step is based on the reaction between quinolones and terbium(III) to form luminescent chelates, which were determined at lambda(ex) 340 and lambda(em) 545 nm (FL mode) or at lambda(ex) 281 and lambda(em) 545 nm (TR mode). Dynamic ranges of the calibration graphs, obtained with standard solutions of analytes and FL and TR modes, respectively, were 190-3500 and 316-2000 ng mL-1 for marbofloxacin, 8-3500 and 8.1-1500 ng mL-1 for ciprofloxacin, 6.2-3500 and 13-1500 ng mL-1 for danofloxacin, 7.4-3500 and 8.4-1500 ng mL-1 for enrofloxacin, 14-3500 and 20-2000 ng mL-1 for sarafloxacin, 12.5-3500 and 13.9-1200 ng mL-1 for difloxacin, 7.6-3500 and 13-3000 ng mL-1 for oxolinic acid, and 9-2000 and 130-3000 ng mL-1 for flumequine. Limit of detection values obtained using FL and TR modes, respectively, were 60 and 95 ng mL-1 for marbofloxacin, 2 and 2.4 ng mL-1 for ciprofloxacin, 1.9 and 3.9 ng mL-1 for danofloxacin, 2.2 and 2.5 ng mL-1 for enrofloxacin, 3.8 and 7 ng mL-1 for sarafloxacin, 4 and 4.2 ng mL-1 for difloxacin, 2.3 and 4 ng mL-1 for oxolinic acid, and 2.7 and 40 ng mL-1 for flumequine. The precision was established at two concentration levels of each analyte and expressed as the percentage of relative standard deviation with values ranging between 1.9 and 7.8%. The validation procedure for the analysis of samples was carried out using European Community recommendations, and the decision limit and detection capability were calculated for bovine whole milk. The method was applied to whole, semiskimmed, and skimmed milk samples spiked with the target analytes, and the recoveries ranged between 93.3 and 106.0%.     

Molecularly imprinted polymer online solid-phase extraction coupled with high-performance liquid chromatography-UV for the determination of three sulfonamides in pork and chicken

A selective imprinted amino-functionalized silica gel sorbent was prepared by combining a surface molecular imprinting technique with a sol-gel process for online solid-phase extraction-HPLC determination of three trace sulfonamides in pork and chicken muscle. The imprinted functionalized silica gel sorbent exhibited selectivity and fast kinetics for the adsorption and desorption of sulfonamides. With a sample loading flow rate of 4 mL min (-1) for 12.5 min, enhancement factors and detection limits for three sulfonamides ( S/ N = 3) were achieved. The precision (RSD) for nine replicate online sorbent extractions of 5 microg L (-1) sulfonamides was less than 4.5%. The sorbent also offered good linearity ( r (2) > 0.99) for online solid-phase extraction of trace levels of sulfonamides. The method was applied to the determination of sulfonamides in pork and chicken muscle samples. The prepared polymer sorbent shows promise for online solid-phase extraction for HPLC determination of trace levels of sulfonamides in pork and chicken samples.