Development of a homogeneous immunoassay based on the AlphaLISA method for the detection of chloramphenicol in milk, honey and eggs

BACKGROUND: A homogenous light‐induced chemiluminescence immunoassay was developed using AlphaLISA technology for the detection of chloramphenicol (CAP). This technology is based on two different kinds of bead, namely light‐sensitive donor beads and beads containing chemiluminescers, also called acceptor beads. A competitive CAP AlphaLISA method was established using artificial antigen‐coated acceptor beads, polyclonal antibodies, biotinylated goat anti‐rabbit IgG and streptavidin‐coated donor beads. RESULTS: The sensitivity of detection was 0.0086 ng mL^?1 and the working range was from 0.0096 to 25 ng mL^?1. The intra‐ and inter‐assay coefficients of variation were both below 10%. The average recovery rates at spiked levels of 0.05–10 ng mL^?1 were 103.2, 108.4 and 91.6% for milk, honey and eggs respectively. The data obtained from the samples showed good correlation with ELISA results. CONCLUSION: The CAP AlphaLISA method is highly sensitive, specific and rapid and is suitable for screening large quantities of samples. Copyright © 2012 Society of Chemical Industry     

免疫亲和柱-高效液相色谱法测定牛奶中氯霉素和玉米赤霉醇及其类似物

建立免疫亲和柱同时净化-高效液相色谱法测定牛奶中氯霉素和玉米赤霉醇及其类似物（α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮和玉米赤霉烯酮）残留量的方法。样品经免疫亲和柱净化与富集后,用高效液相色谱-紫外检测器检测。采用Cloversil-C18反相柱分离,流动相为乙腈-甲醇-水溶液,检测波长为265 nm。结果表明,牛奶中添加氯霉素和玉米赤霉醇及其类似物的回收率在74%～101%,且相对标准偏差均小于7%。氯霉素的检出限为0.02 μg/L,玉米赤霉醇及其类似物的检出限分别为α-玉米赤霉醇0.03 μg/L、β-玉米赤霉醇0.03 μg/L、α-玉米赤霉烯醇0.03 μg/L、β-玉米赤霉烯醇0.03 μg/L、玉米赤霉酮0.04 μg/L和玉米赤霉烯酮0.05 μg/L。该方法灵敏度高、重复性好,提高了检测速率,可满足牛奶样品中痕量氯霉素和玉米赤霉醇及其类似物残留的测定。     

Quantitative Determination of Deoxynivalenol (DON) Using the Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA)

An indirect competitive Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA) was established by using anti-deoxynivalenol (DON) polyclonal antibody and coating antigen DON-bovine serum albumin (BSA) for detection of DON in cereals. DON-BSA was coated on Acceptor bead, the kit also contains donor bead sensitizer particles coated with streptomycin. The optional test conditions and analytical performance of the method were studied. Working concentration ranged from 0.007 to 100 ng/ml and the sensitivity for detection was 0.007 ng/ml. The intra- and inter-batch coefficient of variation (CV) of the assay were both below 5%. The recovery rate of artificially contaminated cereals ranged from 81.1% to 110.9% while the mean recovery of DON from cereals was 94.4%. This study suggests that DON-AlphaLISA method is a good method with high sensitivity and rapidity for quantitative analyzing DON in cereals.     

A Novel Method for Bisphenol A Analysis in Dairy Products Using Graphene as an Adsorbent for Solid Phase Extraction Followed by Ion Chromatography

A novel and reliable ion chromatography (IC) method using graphene (G) as a solid phase extraction (SPE) adsorbent for the rapid analysis of bisphenol A (BPA) in dairy products was developed. The performances of graphene (G) and commercial C18 for BPA extraction from dairy samples were evaluated; results showed that G had higher adsorption efficiency. IC coupled with an electrochemical detector (ED) is eco-friendly, labor and time saving compared to liquid chromatography mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS). The effects of the experimental parameters of the IC-ED system were assessed, and the parameters were optimized to provide maximum sensitivity. The linear range is 5–20,000 ng?mL^?1 with an R value of 0.999. The limit of the detection is 0.8 ng?mL^?1 for a 25-μL injection loop. The mean relative recoveries ranged between 83.3 % and 104.6 %, the corresponding inter-day precision was below 5.3 % for 20, 200, 2,000, and 15,000 ng?mL^?1. This method was successfully employed to analyze BPA in dairy samples.     

A multi-residue TLC screening procedure for anabolic oestrogens and detection of oestradiol, DES or zeranol in chicken muscle tissue extracts

A multi-residue HETLC (High Efficiency Thin Layer Chromatography) screening procedure for 17 beta-oestradiol, diethylstilboesterol (DES), zearalanol (zeranol), zearalenone and their metabolites oestrone, zearalanone, and zearalenol is described. The anabolic oestrogens were analyzed on HETLC plates coated with silica gel and were developed in methylene chloride:methanol: 2-propanol (97:1:2 v/v). The spots were visualized by exposure to iodine vapours and subsequently sprayed with 1% starch solution. Analysis of standards by HETLC at 4 degrees C as a seven-component mixture showed six discrete bands with mean Rfs of 0.37 (oestrone), 0.35 (zearalanone and zearalenone), 0.26 (t-DES), 0.23 (oestradiol), 0.17 (zearalenol and zearalanol), and 0.15 (c-DES). Chicken muscle tissues (1, 2.5, or 5 g) were extracted with 95% acetone. Extracts were then fortified with 50-250 ng each of the anabolic oestrogens, purified in alumina and ion-exchange columns and analyzed by HETLC. Oestradiol, zeranol or DES in fortified tissue extracts were clearly detected when an equivalent of 4 ng were analyzed by HETLC after purification in alumina and ion-exchange columns. The intensity of their bands suggested near quantitative recovery when compared to intensity of bands of known amounts of standards. The described extraction, purification, and TLC procedures can be used to screen these oestrogens at low ppb amounts in chicken muscle tissues and should be applicable to screen tissues of cattle and sheep.     

Development of an immunochromatographic assay for the β-adrenergic agonist feed additive zilpaterol

Zilpaterol is a β-adrenergic agonist feed additive approved in the United States to increase weight gain and improve feed efficiency of cattle. A zilpaterol immunochromatographic assay was developed as an economical and user-friendly rapid detection method for zilpaterol and validated using urine and tissue samples derived from animal studies. The assay sensitivity was 1.7–23.2 ng g^?1 or mL^?1 across a variety of feed and animal matrices and did not cross-react with clenbuterol or ractopamine. No sample pre-treatment of cattle and sheep urine was needed, but horse urine and feed required dilution; skeletal muscle required solvent extraction prior to testing. Of 32 incurred sheep urine samples tested, zilpaterol content was correctly identified in all but 2 samples. Horse urine containing >10 ng mL^?1 of incurred zilpaterol residue (n = 48) was correctly identified as zilpaterol positive. The assay correctly identified 0-day withdrawal sheep muscle samples as zilpaterol positive and the control and longer withdrawal day sheep muscle samples as negative. Zilpaterol was demonstrated to be stable in horse urine when stored at ?20°C for 7 years.     

Caffeine Content of Retail Market Coffee in Portugal

How much caffeine does one ingest when drinking a simple cup of coffee in Portugal? The study presented herein tried to answer this question through the assessment of caffeine content of commercially available espresso coffee samples, both caffeinated and decaffeinated, using a high-performance liquid chromatography assay. Caffeine was rapidly separated from the sample matrix using a RP-18 column (250?×?4 mm i.d., 5 μm). The flow rate was 1.0 mL/min and the mobile phase consisted of water acidified with 5% of orthophosphoric acid/methanol (35:65, v/v). Caffeine was detected directly at 273 nm. The assay was validated for linearity, lower limit of quantification and limit of detection, precision, accuracy, and stability. Seventeen different brands of caffeinated coffee and six of decaffeinated coffee were analyzed. As for capsule coffee, eight caffeinated and two decaffeinated blends were analyzed. The caffeine content of caffeinated coffee varied from 53.8?±?5.9 to 141.3?±?5.3 mg/cup, whereas for caffeinated capsule coffee caffeine concentrations ranged from 45.0?±?5.3 to 60.8?±?6.2 mg/cup. As for decaffeinated coffee, caffeine concentrations ranged from 0.96?±?0.04 to 3.9?±?0.1 mg/cup and for decaffeinated capsule coffee from 0.93?±?0.04 to 1.2?±?0.1 mg/cup.     

A Preconcentration Procedure for Determination of Ultra-Trace Mercury (II) in Environmental Samples Employing Continuous-Flow Cold Vapor Atomic Absorption Spectrometry

New functionalized magnetic nanoparticles as solid-phase sorbent were prepared and investigated for extraction of ultra-trace amounts of mercury from environmental samples. The Fe₃O₄ magnetic nanoparticles functionalized with dithizone were characterized by Fourier transform infrared spectrometer. X-ray diffraction and scanning electron microscopy confirmed the size of nanoparticles. Effects of several factors on the extraction procedure were investigated. The optimized conditions were established to be 80 mg of polymer, 8.5 for solution pH, 5 min for adsorption time, 5 min for desorption time, 2 mL for HCl (0.1 mol L^?1)/ thiourea 0.05 % as the eluent, 500 mL for breakthrough volume, and without addition of salt. Under the optimal conditions, the limit of detection, maximum capacity, and preconcentration factor were 0.05 ng mL^?1, 0.557 mmol g^?1, and 250, respectively. Limit of quantification was in the range of 0.2–2 ng mL^?1 for various matrices. Accuracy and precision of the method were about ±2.0 and below 11.1 %, respectively. Finally, the present method has been successfully applied to mercury determination in table salt, green tea, vegetables, toothpaste, and water samples. The mercury content found in the real samples was from 0.6 to 15.74 ng mL^?1 without addition of mercury.     

Determination of chloramphenicol and zeranols in pig muscle by immunoaffinity column clean-up and LC-MS/MS analysis

An immunoaffinity column clean-up and LC-MS/MS method was successfully developed for simultaneous determination of chloramphenicol, zearalanone, α-zearalanol, β-zearalanol, zearalenone, α-zearalenol and β-zearalenol in pig muscle. The sample was extracted with diethyl ether after enzymatic digestion by β-glucuronidase/sulfatase. The extracted solution was evaporated to dryness and the residue was then dissolved in 1 ml of 50% acetonitrile solution. After filtration and dilution with phosphate buffer solution (PBS), the reconstituted solution was cleaned-up with an IAC-CZ immunoaffinity column and then analysed by HPLC-MS/MS. The established method were validated by linearity (r ≥ 0.9990), precision (RSD ≥ 2.9%), average recovery (74.5–105.0%) and limit of detection (0.04–0.10 μg kg^–1). The developed method is rapid, reliable, sensitive, accurate and has good applicability for real samples.     

Development of an Immunochromatographic Test Strip for Rapid Simultaneous Detection of Enrofloxacin and Ofloxacin in Tissue of Chicken Muscle and Pork

ENR and OFL are the most consumed quinolones on livestock in China. In this work, we developed a rapid immunochromatographic lateral flow test strip for simultaneous detection of the residues of enrofloxacin and ofloxacin in chicken muscle and pork. We screened an anti-ENR and OFL monoclonal antibody. The IC₅₀ of anti-ENR and anti-OFL were 6.67 and 7.13 ng/ml, respectively. The present immunochromatographic lateral flow test strip is a one-step assay and required much less professional personnel and experimental instruments. In the present study, the decision limit (CCα) of the test strip was calculated to be 0.089 ng/mL and detection capability (CCβ) was 0.217 ng/mL with the scanner. The limit of detection was estimated to be 10 ng/mL. According to parallel HPLC analysis with 47 blind samples, coincidence rate is 100 % when the contents of ENR and OFL were more than 10 ng/mL. Results indicated that the strip test we developed had good reliability, and the strip test gave neither false positive nor false negative results. It will provide results within 20 min without special equipment. Therefore, the test strip is very useful as a screening method for semi-quantitative or qualitative detection of enrofloxacin and ofloxacin in chicken muscle and pork.     

Simultaneous Determination of Ractopamine,Chloramphenicol, and Zeranols in Animal-Originated Foods by LC-MS/MS Analysis with Immunoaffinity Clean-up Column

A novel analytical method employing immunoaffinity column (IAC) clean-up coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of ractopamine, chloramphenicol, and zeranols (α-zearalanol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol, and zearalenone) in animal-originated foods. The sample was first digested by β-glucuronidase/sulfatase and then extracted with ethyl acetate-diethyl ether (9:1, v/v). The extracted solution was evaporated to dryness and then the residue was dissolved by 2 mL of 50% acetonitrile solution. After filtration, 1 mL filtrate was diluted to 10 mL with PBS. The reconstituted solution was cleaned up with immunoaffinity column and then analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The established method was shown to be sensitive efficient and reliable as indicated by the linearity (r ² ≥ 0.9994), precision (RSD ≤ 1.7%), average recovery (72.3–103.2%), and the limit of detection (0.05–0.10 μg/kg). The method can be used for determination of trace residues of ractopamine, chloramphenicol, and zeranols in animal-originated foods.     

Development of a sensitive polyclonal antibody-based competitive indirect ELISA for determination of citrinin in grain-based foods

ABSTRACT

A sensitive competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for the detection and quantification of citrinin (CIT) in grain-based food samples. The limit of quantification (IC₂₀) of the established method was 0.10 ± 0.02 ng mL^?1, with the limit of detection (IC₁₀) being 0.04 ± 0.007 ng mL^?1 in wheat and corn flour matrices with a coefficient of variation (CV) less than 20%. The assay was very specific to CIT and showed no cross-reactivity with other mycotoxins (OTA, T-2 toxin, HT-2 toxin, DON, patulin and zearalenone). In spiked wheat and corn flours, the recoveries ranged from 86.6% to 115.6% with CVs of less than 20%. The effectiveness of this method was verified by participating in a proficiency test (PT) from the Food Analysis Performance Assessment Scheme (FAPAS) 17181 corn flour. A successful z-score (?0.6) for this PT sample showed that the present method is comparable to the instrumental methods used by other laboratories in the PT testing scheme. A small survey of grain-based foods was conducted using this method and CIT was detected in 43% of the samples up to a concentration of 17.7 ng g^?1. This method is suitable for sensitive and rapid quantitation of citrinin in wheat and corn matrices.     

A New Extraction Method for the Determination of Oxytocin in Milk by Enzyme Immune Assay or High-Performance Liquid Chromatography: Validation by Liquid Chromatography–Mass Spectrometry

There has been a controversy regarding the use of exogenous oxytocin (OT) in milking cattle which may have toxicological consequences during nonphysiological exposure. In the present study, a new sensitive extraction method for OT was developed followed by enzyme immune assay (EIA) or high-performance liquid chromatography (HPLC) analysis. The extraction of OT in milk involves two steps: (1) TCA precipitation of milk proteins and (2) solid-phase extraction (SPE) cleanup process. Without these steps, analysis of OT in milk was not possible. Utilizing EIA as a quantitative tool the limit of detection (LOD) and limit of quantitation (LOQ) were found to be 7.74 and 10.3 pg?ml^?1, precision in terms of intra- and interday coefficient of variation was below 13 % (%RSD, N?=?8), while percent recoveries were between 85 and 92 %. Utilizing UV-HPLC, the LOD, LOQ, precision, and recovery values were found to be 4.1 ng?ml^?1, 9.8 ng?ml^?1, 2–10 %, and 84–91 %, respectively. OT was found to be stable against adverse temperature (up to 100 °C) and pH (2 to 10) and simulated gastric fluid digestibility assay. Four milk samples collected from the market were analyzed, which showed that TCA precipitation and SPE steps are mandatory and the results were validated by LC-MS showing mass ion peak at 1 kD.     

A Sensitive Immunoaffinity Column-Linked Indirect Competitive ELISA for Ochratoxin A in Cereal and Oil Products Based on a New Monoclonal Antibody

A new sensitive monoclonal antibody (mAb) 1H2 against ochratoxin A (OTA) was reported herein. This mAb belonged to the immunoglobulin G1 (k chain) isotype. In the optimized indirect competitive enzyme-linked immunosorbent assay (icELISA), 1H2 showed a 50 % inhibition concentration (IC₅₀) value of 0.058 ng/mL and a detection limit (IC₁₀) of 0.001 ng/mL. The cross-reactivity of 1H2 with ochratoxin B, aflatoxins, deoxynivalenol, zearalenone, T-2 toxin, or fumonisins was below 0.3 %. Based on this mAb, an immunoaffinity column (IAC)-linked icELISA was developed for OTA detection in the cereal and oil products. The working range of the assay for solid sample was 0.36–16 μg/kg. The recoveries from spiked samples of IAC-linked icELISA ranged from 83 to 101 %. These recoveries were much higher than those of icELISA (21–78 %) and in good agreement with those obtained by using the standard high-performance liquid chromatography method (87–110 %). The results indicated that the mAb 1H2 had the values for studies of OTA in the crude agricultural products.     

Development of a screening analytical method for the determination of non-dioxin-like polychlorinated biphenyls in chicken eggs by gaschromatography and electron capture detection

ABSTRACT

A sensitive and reproducible screening analytical method is here proposed for the determination of six non dioxin-like polychlorinated biphenyls (NDL-PCBs, congener 28, 52, 101, 138, 153, 180) in chicken eggs based on accelerated solvent extraction (ASE) procedure for the fat extraction and determination, a solid phase extraction (SPE) sample clean-up process, and a gas chromatography – electron capture detection (GC-ECD) analysis. The optimized chromatographic separation, in less than 25 min, returned good responses for the six NDL-PCBs in the range of 2.5–60.0 µg L^?1, with correlation coefficients always higher than 0.9995. Instrumental limits of detection were between 0.08–0.35 µg L^?1, corresponding to 0.05 and 0.23 ng g^?1 fat in the matrix, while method detection limits, calculated on spiked egg samples, ranged from 1.6 to 3.5 ng g^?1 fat. The method has been extensively validated in terms of selectivity, sensitivity, recovery, precision, ruggedness and measurement uncertainty, following the European Directives.     

Validated RP-HPLC Method for Simultaneous Determination of Tocopherols and Tocotrienols in Whole Grain Barley Using Matrix Solid-Phase Dispersion

The vitamers of vitamin E such as α-, β-, γ-, and δ-tocotrienol and α-, β-, γ-, and δ- tocopherol are important phytochemical compounds with antioxidant activity and with potential benefits for human health. A high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was validated for their determination in whole grain barley samples. Tocol extraction was performed by an optimized matrix solid-phase dispersion (MSPD) protocol with neutral alumina (0.5 g) as the dispersion agent and methanol (5 mL) as the elution solvent. The analytical column was an Eclipse XDB C18 column (150?×?4.6 mm, 5 μm) and it was operated at room temperature. Mobile phase was consisted of methanol/acetonitrile/i-propanol (55:40:5?v/v?%) and the elution was isocratic at a flow rate of 0.8 mL/min. Total analysis time was 12 min, and the detection of the tocols was performed with a fluorimetric detector where the excitation and emission wavelengths were set at 295 and 335 nm, respectively. Method validation was performed by means of intra-day (n?=?5) and inter-day accuracy and precision (n?=?8), sensitivity, and linearity. The linear regression coefficient (R ²) was higher than 0.99. The recoveries of the tocols from barley samples with the proposed extraction method were in an acceptable level (74–91 %) where the relative standard deviation ranged from 4.2 to 15.0 %. Limits of detection (LODs) and limits of quantification (LOQs) varied from 0.03 to 0.11 mg kg^?1 and 0.11 to 0.34 mg kg^?1, respectively.     

LC-MS/MS Determination of Organophosphorus Pesticide Residues in Coconut Water

A simple and specific analytical method based on solid phase extraction (SPE), and liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) operated in multipule reaction monitoring (MRM) mode with dynamic time segment acquisition windows, was developed to determine organophosphorus pesticide residues (acephate, monocrotophos, dimethoate, malaoxon, dichlorvos, malathion, phenthoate, parathion-ethyl, chlorfenvinfos, quinalphos, diazinon, phosalone, profenofos, ethion, chlorpyrifos) in tender coconut water. The method was observed to be linear with lower limit of detections ranged from 0.1 to 1.5 ng/mL (measured at S/N ratio 5:1) and limit of qunatitation 0.5 to 2.0 ng/mL (measured at S/N ratio 10:1) for all studied pesticides. The intra- and inter-day assay accuracies calculated at four concentration levels in six replicates (n?=?6) were in the range of 80 % to 115 % with relative standard deviations (RSD) below 15 %. The percentage recoveries of all the analysed pesticides were in the range of 86.8 % to 107.6 % and the calculated RSD values were below 12 %. The developed method was applied to determine pesticide residues in natural tender coconut water samples. The results showed the presence of malathion ranging from 24 to 45 ng/L concentrations in some natural tender coconut water samples collected from various local market places in and around Hyderabad, Andhra Pradesh, India.     

Slurry Sampling and HG AFS for the Determination of Total Arsenic in Rice Samples

This paper proposes a procedure for the determination of total arsenic in rice using slurry sampling and hydride generation atomic fluorescence spectrometry (HG AFS). During optimization, water, hydrochloric acid, and nitric acid solutions were tested as extractor. Best results were found using 2.0 mol?L^?1 nitric acid solutions. The slurries are prepared using 200 mg of rice sample, 5.0 mL of nitric acid solution, and sonication for 30 min. Then, an aliquot of 5.0 mL of the slurry was taken and 3.0 mL of 1.5 mol?L^?1 hydrochloric acid solution and 1.5 mL of 10 % potassium iodide in 2 % ascorbic acid solution were added. After 30 min, the volume was completed up to 10 mL with ultra-pure water and arsenic quantified by HG AFS. In these experimental conditions, the method allows the determination of arsenic employing external calibration, with limits of detection and quantification of 1.1 and 3.3 ng?g^?1 and precision expressed as relative standard deviation (%RSD) varying between 5.9 and 1.3 % for arsenic concentration of 0.12 and 0.47 μg?g^?1. Accuracy was confirmed by analysis of the NIES standard reference material for rice flour, SRM 10b. This method was used to determine the arsenic content in 20 rice samples that were purchased at supermarkets in Salvador, Bahia, Brazil. The arsenic content for the three types of rice (white, parboiled, and brown) varied from 0.12 and 0.47 μg?g^?1. Some of the samples were also analyzed by HG AFS after complete mineralization in block digester employing a reflux system (cold finger). A statistical test showed that there was no significant difference between the results obtained using slurry sampling and those obtained after complete digestion of the sample.     

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of sulfonamides,trimethoprim and dapsone in muscle,egg, milk and honey

A quantitative multi-residue method that includes 13 sulfonamides, trimethoprim and dapsone was developed and validated according to Commission Decision 2002/657/EC for muscle, milk egg and honey samples. For all matrices, the same extraction procedure was used. Samples were extracted with an acetone/dichloromethane mixture and cleaned up on aromatic sulfonic acid (SO₃H) SPE cartridges. After elution and concentration steps, analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data were acquired according to the multiple reaction-monitoring approach (MRM) and analytes were quantified both by the isotope dilution and the matrix-matched approaches calculating the response factors for the scanned product ions. The developed method shows good linearity, specificity, precision (repeatability and within-laboratory reproducibility), and trueness. Estimated CCβ for sulfonamides ranged between 5.6 and 8.2 µg kg^?1 for eggs, between 11.1 and 69.9 µg kg^?1 for milk, between 64.7 and 87.9 µg kg^?1 for muscle, and between 2.7 and 5.3 µg kg^?1 for honey. CCβ values for dapsone were 3.1, 0.6, 0.7 and 1.5 µg kg^?1 and for trimethoprim were 3.1, 6.7, 81.7 and 3.0 µg kg^?1 calculated for eggs, milk, muscle and honey, respectively. Recovery for all matrices was in the range from 89.1% and 109.7%. In matrix effect testing, no significant deviations were found between different samples of muscle and milk; however, a matrix effect was observed when testing different types of honey. The validation results demonstrate that the method is suitable for routine veterinary drug analysis and confirmation of suspect samples.     

Determination of 21 Free Amino Acids in Fruit Juices by HPLC Using a Modification of the 6-Aminoquinolyl-<Emphasis Type="Italic">N</Emphasis>-hydroxysuccinimidyl Carbamate (AQC) Method

The present paper reports the application of an optimized pre-column derivatization procedure with aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to the determination of 21 free amino acids in fruit juices. The method permitted the optimal separation of 21 amino acids and ammonium in 30 min. Excellent response linearity was obtained from 2 to 1,000 μM for all amino acids, except tryptophan, for which the linearity was 5–1,000 μM. The fluorescence detection limits ranged from 0.08 pmol (phenylalanine) to 0.69 pmol (cysteine), except for tryptophan (7.89 pmol), for a signal-to-noise ratio of 3. The relative standard deviations of peak areas ranged from 0.1 to 0.4 % for intraday analysis and from 2.1 to 5.0 % for interday analysis. The method was applied to analyze the free amino acids in six fruit juices, and the recoveries of the proposed method were 90.4 to 103.3 %. The total free amino acid content ranged from 56.97 mg L^?1 (apple) to 469.45 mg L^?1 (longan) in six fruit juices. Aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), and glutamine (Gln) were present in higher levels in most samples, whereas cysteine (Cys) was below the detection limit in the selected samples. Notable advantages of this method include its speed, ease, low cost, good repeatability, high sensitivity, excellent precision and accuracy, and anti-interference in high-sugar matrices.