Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Difenoconazole Residues in Fruits and Vegetables

An indirect, competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of difenoconazole was developed. Two haptens were designed and successfully synthesized. Hapten 1 had a particular moiety of difenoconazole, while hapten 2 had its full structure. The polyclonal antibodies against hapten-protein conjugates were prepared by immunizing rabbits. After optimization of the conditions, the detection limit (IC₁₅) and sensitivity (IC₅₀) were 4.58 and 29.10 μg L^?1, respectively. The cross-reactivities of the antibody with 11 triazole fungicides were all less than 0.1%, which showed that the antibody had excellent specificity. The recoveries of difenoconazole from the spiked samples ranged from 89.70 to 102.31% with good accuracy. The matrix effect was easily removed using a simple, rapid, and efficient extraction method on fruits and vegetables. The detection limit was all 229 μg kg^?1 in fruits and vegetables. To validate the ic-ELISA, samples were spiked with difenoconazole at three different concentrations and simultaneously analyzed using high-performance liquid chromatography (HPLC). The results showed a good correlation between the ic-ELISA and HPLC data (R ² = 0.9970). As a result, the developed immunosorbent assay is suitable for the quantitative determination of difenoconazole in fruits and vegetables.     

酶联免疫吸附法检测动物源性食品中恩诺沙星残留量

为检测动物源性食品中恩诺沙星残留量,评估基于卵黄抗体的间接竞争酶联免疫吸附法检测恩诺沙星的可行性,用活性脂法将恩诺沙星同卵清蛋白偶联制备免疫原和包被抗原并用紫外光谱进行验证。用聚乙二醇-6000提取卵黄抗体。五免之后效价达到峰值1∶32 000。用间接竞争酶联免疫吸附法确定包被原质量浓度、卵黄抗体的稀释倍数和IC_(50)分别为38 ng/m L、1∶64 000和18.207 ng/m L,回归曲线方程为y=0.891 1-0.016 5x(R~2=0.990)。结果表明,制备的抗恩诺沙星卵黄抗体为进一步建立检测动物源性食品中恩诺沙星的残留的免疫方法提供参考依据。     

Optimization of Extraction Parameters of Total Phenolics from <Emphasis Type="Italic">Annona crassiflora</Emphasis> Mart. (Araticum) Fruits Using Response Surface Methodology

In this study, response surface methodology was used to optimize the extraction temperature (25–75 °C) and ethanol concentration (0–70 %, ethanol/water, v/v) to maximize the extraction of total phenolic compounds (TPC) from araticum pulp. The efficiency of the extraction process was monitored over time, and equilibrium conditions were reached between 60–90 min. A second-order polynomial model was adequately fit to the experimental data with an adjusted R ² of 0.9793 (p < 0.0001) showing that the model could efficiently predict the TPC content. Optimum extraction conditions were ethanol concentration of 46 % (v/v), extraction temperature of 75 °C and extraction time of 90 min. Under the optimum conditions, the araticum pulp showed high TPC content (4.67 g GAE/100 g dw) and also high antioxidant activity in the different assays used (46.56 μg/mL, 683.65 μmol TE/g and 1593.72 μmol TE/g for DPPH IC₅₀, TEAC and T-ORAC_FL, respectively). From our extraction procedure, we successfully recovered a significantly higher amount of TPC compared to other studies in the literature to date (1.5–22-fold higher). Furthermore, TPC and antioxidant activity were present in the fruit in levels that are difficult to find in other common fruits. These results expose a potential approach for improving human health through consumption of araticum fruit.     

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 Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors

A phage display library of single chain variable fragment (scFv) antibodies was constructed to screen specific scFv antibodies against amatoxins. One recombinant scFv antibody with high specificity for amatoxins, termed scFv-A4, was isolated from the library by biopanning. SDS-PAGE analysis showed that the soluble scFv-A4 protein was expressed successfully, and the protein posed relatively good specificity with an IC₅₀ of 77.48 ng/mL and IC₁₅ of 1.91 ng/mL using indirect competitive ELISA (ic-ELISA). On the basis of the expressed scFv-A4 protein, a rapid and simple new immunoassay using surface plasmon resonance (SPR) sensor for the detection of amatoxins was developed. The IC₅₀ and IC₁₅ of new immunoassay were 7.66 and 0.17 ng/mL. The sensitivity of immunoassay using SPR sensor was about 10 times that of ic-ELISA. These results showed the potential usefulness and advantages of new immunoassay using SPR sensor for the detection of toxins.     

Preparation of a Broadly Specific Monoclonal Antibody-Based Indirect Competitive ELISA for the Detection of Benzodiazepines in Edible Animal Tissues and Feed

Incorrect use of benzodiazepines could result in serious health problems. To monitor the illegal use of benzodiazepine compounds in animals, a group-specific monoclonal antibody (mAb) was prepared in this study. The obtained 3D7 mAb, which is an IgG1 isotype mAb, displayed an IC₅₀ value of 8.9 ng mL^?1 for diazepam and exhibited cross-reaction for diazepam (100 %), nitrazepam (49 %), nordiazepam (140 %), temazepam (32 %), oxazepam (17 %), estazolam (7.5 %), and alprazolam (2.4 %). Based on this mAb, for the first time in this study, an optimized indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) protocol that did not require complicated sample preparation and clean-up was developed. The detection limits of this ic-ELISA for benzodiazepines ranged from 1.2 to 3.3 μg kg^?1 in muscle matrix and from 25.2 to 55.4 μg kg^?1 in feed matrix. The recoveries ranged from 70.9 to 111.3 % with coefficients of variation below 15.0 %. Good correlations (r?>?0.9494) between the results of the ic-ELISA and high-performance liquid chromatography were also observed. This simple method reduced the time required for sample preparation, ensured greater throughput, and met the requirements for benzodiazepine residue analyses. In conclusion, the proposed method is a sensitive and rapid multi-residue technique that offers a cost-effective alternative to current published procedures without any concession in the ability to detect benzodiazepine sedative misuse.     

Development of an Indirect Competitive ELISA Kit for the Rapid Detection of Benzopyrene Residues

This paper presents the generation of monoclonal antibodies (mAbs) with high specificity against benzopyrene (BaP) through cell fusion procedures, and the development of mAb-based indirect competitive enzyme-linked immunoabsorbent kit (icELISA kit) to detect BaP using one of these hybridomas (clone 2E12). The calibration curve of BaP-Kit with standard BaP inhibitor was typical sigmoid curve fitted to the four parameters logistic equation with the linear regression equation y?=??28.662x?+?132.87, the half-maximal inhibition concentration (IC₅₀) of 0.779 μg/L and the sensitivity of 0.054 μg/L. The recovery of BaP spiked in beef was between 81.3 and 93.6 %. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 6 %. Except for minor cross-reactivity with benzo[b]fluoranthene (2.7 %), other interference to the assay was negligible (<0.05 %). The validity of BaP kit in 4 °C was above 6 months. The sensitivity, specificity, simplicity, and reliability of BaP kit were confirmed by the procedure utilizing gas chromatography coupled to mass spectrometric (GC-MS) for the identification and quantification of BaP in barbecue. Their coincidence rate was 100 % compared with GC-MS. The kit was proved to be used for the rapid determination of BaP residues.     

Combination of Thermosonication and Pulsed Electric Fields Treatments for Controlling <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in Chinese Rice Wine

The objective of this study was to investigate the combined effect of thermosonication (TS) and pulsed electric fields (PEF) against Saccharomyces cerevisiae in Chinese rice wine. The effectiveness of standalone TS treatment (35 °C, 750  W, 120 min) on the inactivation of S. cerevisiae was insignificant (0.76 log CFU/mL). However, 2.88 log CFU/mL of S. cerevisiae were inactivated when the standalone PEF treatment with moderate conditions (35 °C, 12 kV/cm, 120 μs) was applied. The combination of TS and PEF had an additive effect on the inactivation of S. cerevisiae, and the sequence applied (TS-PEF or PEF-TS) markedly influenced the inactivation results (P < 0.05). In particular, the microbial inactivation by TS-PEF (3.72 log CFU/mL) was higher than that by PEF-TS (3.48 log CFU/mL); this result indicates that PEF were able to restrain the effect of TS. On the other hand, TEM micrographs of S. cerevisiae after the different treatments showed that the combined techniques resulted in more severe disruptions on cells. Higher cytoplasmic shrinkage and more intracellular material leakage were observed from the TEM observations of the cells treated by TS-PEF. These results may serve as a reference of the potential application of the combined treatment TS-PEF for microbial inactivation in Chinese rice wine.     

Quality Changes in Mango Juice Treated by High-Intensity Pulsed Electric Fields Throughout the Storage

The effect of high-intensity pulsed electric fields (HIPEF) processes on Listeria innocua inhibition, physicochemical parameters and activity of oxidative enzymes of mango juice was evaluated to set the optimal HIPEF treatment time. Quality parameters, microbial population and bioactive compounds of HIPEF-treated (35 kV/cm, 1800 μs) and thermally treated (TT) (90 °C, 60 s) mango juices were studied and compared with those non-treated during 75 days of storage at 4 °C. HIPEF treatment for 800 μs ensured 5 log reductions of L. innocua. Polyphenoloxidase (PPO), lipoxygenase (LOX) and peroxidase (POD) residual activities were significantly reduced to 70, 53 and 44%, respectively, at treatment times of 1800 μs. Similar sensory properties compared with fresh mango juice were attained from product treated at 1800 μs. Moreover, fresh mango juice colour (L* = 38.79, h° = 106.57) was preserved after HIPEF treatment throughout storage. Moulds and yeasts and psychrophilic bacteria counts in HIPEF-treated (1800 μs) mango juice remained below 6 log cycles CFU/mL up to 2 months of refrigerated storage. The content of total phenolic compounds in those HIPEF-treated increased from 333 to 683 μg of GAE/mL from day 0 to the end of storage. Hence, the application of HIPEF may be a feasible treatment in order to ensure microbiological stability, high bioactive compound content and fresh-like characteristics of mango juice.     

Analysis of Ascorbic Acid and Isoascorbic Acid in Orange and Guava Fruit Juices Distributed in Thailand by LC-IT-MS/MS

This work describes the determination of ascorbic acid (AA) and isoascorbic acid (IAA) in fruit juices by liquid chromatography coupled with ion trap tandem mass spectrometry (LC-IT-MS/MS), with a reversed phase column (C₁₈) and simple isocratic conditions of 0.1 % formic acid. The negative ion mode of electrospray ionization (ESI) and the MS/MS transition of m/z 175 were used for the quantitation of AA- and IAA-generated product ions with m/z 115. The method was validated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy. Good linearity was achieved with 0.6 and 1.8 μg/mL for the LOD and LOQ, respectively. The intraday and intermediate precisions were approximately 4 and 7 %, respectively. Recovery percentages ranged from 88 to 108 %. All validation parameters were found to be within the acceptable range for both AA and IAA. Hence, the proposed method is suitable for simultaneously determining AA and IAA. Fourteen fruit juice samples were analyzed including fruit juices from supermarkets, local markets, and laboratory preparations. Ten fruit juice samples (100 %) from different brands in the supermarkets were investigated for AA and IAA content. AA could be detected in all the samples, ranging from 5.2?±?2 to 44.4?±?1.3 μg/mL, and the values of vitamin C in 4 guava samples were less than the values specified by the manufacturer. IAA was observed in 4 of 10 samples ranging from 3.4?±?0.9 to 78.3?±?3.9 μg/mL, and the highest value of IAA was approximately two fold higher than the highest value of AA. For freshly prepared fruit juices, AA was detected in both local market- and laboratory-prepared juices. The value ranged from 13.2?±?0.9 to 105.1?±?1.3 μg/mL. In addition, the AA stability after opening the package was evaluated. The results showed that after 4 days of storage in the refrigerator, approximately 30 and 15 % losses of AA were observed for the orange and guava juices, respectively. Fresh fruit juices were thus demonstrated to be good sources of AA, with the highest value observed for guava juice prepared in the laboratory.     

Determination of Six Paraben Residues in Fresh-cut Vegetables Using QuEChERS with Multi-walled Carbon Nanotubes and High-Performance Liquid Chromatography–Tandem Mass Spectrometry

In this study, an optimized QuEChERS sample preparation method was developed to analyze residues of six parabens: methyl-, ethyl-, n-propyl-, isopropyl-, n-butyl-, and isobutylparaben in five fresh-cut vegetables (potato, broccoli, carrot, celery, and cabbage) with high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Homogenized samples were extracted using acetonitrile, and the extracts were cleaned with the novel sorbent multi-walled carbon nanotubes (MWCNTs). MWCNTs provided 84–94% removal of chlorophyll and lower matrix effects (MEs) compared to commonly used primary-secondary-amine (PSA) sorbent. Selected parabens were separated by HPLC with isocratic elution using acetonitrile and 0.1% (v/v) formic acid solution and determined by triple quadrupole MS/MS. The method validation results showed that recoveries were at 70–120% with RSDs <20%. Calibration curves showed linear responses of six parabens with R ² > 0.99. Fifty fresh-cut vegetable samples from different farmer markets in Beijing, China were collected to measure the paraben residues, and only one sample was tested positive with methylparaben concentration at 81 μg/kg.     

Influence of Preservative Concentration,pH Value and Fat Content in Raw Milk at Detection Limit of Microbial Inhibitor Tests (Delvotest® Accelerator) for Amoxicillin and Oxytetracycline

The presence of antibiotic residues in raw milk is a big threat not only to the dairy industry but also to public health. Therefore, it is very important to detect antibiotic residues in raw milk. There are many reliable antibiotic test used in dairy laboratories. Unfortunately, some factors can affect the results causing, so-called false-positive results. The aims of this paper are to check the detection limits of microbiological inhibitor test with test organisms Geobacillus stearothermophilus var. calidolactis, which are provided on Delvotest® Accelerator instrument (DSM, The Netherlands) for antibiotics amoxicillin and oxytetracycline and to examine the influence of variations of milk fat contents, pH values and azidiol concentrations in raw milk on detection limits, e.g., test results. The obtained results showed that detection limit for amoxicillin is 3 μg/L and oxytetracycline 250 μg/L; concentration of preservative azidiol of 0.1 mL per 40 mL of milk does not affect the detection limits of the test; microbiological inhibitor test is extremely sensitive to low pH value of samples so, it is recommended to avoid testing milk samples with low pH value, or to adjust the sample’s pH value by addition of some base; and that fat content in raw milk up to 5 % does not affect the detection limit of the methods for amoxicillin and for oxytetracycline.     

A Highly Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) by Antigen Coating for Diethyl Phthalate Analysis in Foods

As we have known, with the plasticizer disturbance in 2011 in Taiwan, long-term exposure to diethyl phthalate (DEP), one of the widely used phthalate esters, can lead to serious health problems. Therefore, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) by antigen-coated plate format for DEP in foods was proposed in this paper. The polyclonal antibodies were raised against diethyl 4-aminophthalate (4-DEAP) conjugated to bovine serum albumin by the amino diazotization linkage method. Coating antigen was prepared with 4-DEAP conjugated to ovalbumin using the same procedure. Under the optimal experimental conditions, the ic-ELISA has a linear working range of 0.005–18.6 ng/mL (R ²?=?0.9921), with a limit of detection of 0.0049 ng/mL. Low cross-reactivity (<9 %) to structurally related phthalates was observed. The method was successfully applied to the determination of DEP in fruit juice, milky tea, pure milk, and sour milk, without purification or preconcentration. Satisfactory recoveries were obtained ranging from 91.1 to 109.3 %. The results suggested that the developed ic-ELISA is a simple, sensitive, and specific method for the rapid monitoring of DEP in food samples.     

Modification of Multiresidue QuEChERS Protocol to Minimize Matrix Effect and Improve Recoveries for Determination of Pesticide Residues in Dried Herbs Followed by GC-MS/MS

An accurate, rapid, and reliable multiresidue QuEChERS method based on gas chromatography coupled with tandem mass spectrometry was developed for the determination of 235 pesticides in challenging, dry, complex herb matrices (Centaurea cyanus L., Matricaria chamomilla L., Thymus vulgaris L.). Sample mass and the type of cleanup sorbent used to estimate the procedure’s effectiveness were optimized. Purification steps with ChloroFiltr, ENVI-Carb, GCB, octadecyl, PSA, and Z-Sep as cleanup sorbents and a step without purification were compared. To minimize the matrix effect and obtain acceptable recoveries for pesticides, 2 g of herb sample and 10 mL acetonitrile, followed by d-SPE cleanup step with a combination of 150 mg PSA/45 mg ENVI-Carb/900 mg MgSO₄, and additionally 50 μL of 5% formic acid and some droplets of dodecane, were needed. Matrix effects for the vast majority of pesticides were reduced (>?20%), showing suppression or enhancement. Most recoveries were in the range of 70–120% (RSD < 18%), reaching the quantification limit of 0.001 to 0.002 mg kg^?1. There was excellent linearity within the range from 0.001 to 2.00 μg mL^?1, and a correlation coefficient higher than 0.999 was obtained. Expanded measurement uncertainty was estimated to be between 4 and 43%. Finally, the developed method was successfully employed to identify and quantify pesticide residues in the analysis of 46 real herb samples.     

A TLC-HPLC Method for Determination of Thiamphenicol in Pig,Chicken, and Fish Feedstuffs

An effective thin-layer chromatography (TLC) purification procedure coupled to high-performance liquid chromatography (HPLC) method was developed for the determination of thiamphenicol (TAP) in pig, chicken, and fish feedstuffs. The feedstuff samples were extracted with ethyl acetate, defatted with n-hexane saturated with acetonitrile, and further purified by TLC. The chromatographic separation was performed on a Waters Symmetry C₁₈ column using an isocratic procedure with acetonitrile-water (21.7:78.3, v/v) at 0.6 mL/min. The ultraviolet (UV) detector was set at a wavelength of 225 nm. The TAP concentrations in feedstuff samples were quantified using a standard curve. Good linear correlations (y?=?162,630x???2381.7, r?>?0.9998) were achieved within the concentration range of 0.05–10.00 μg/mL. The recoveries of TAP spiked at levels of 1, 10, and 100 μg/g ranged from 82.0 to 114.9% with the intra-day and inter-day relative standard deviation (RSD) less than 9.0%. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.03 mg/kg for pig feedstuffs, 0.02 and 0.04 mg/kg for chicken feedstuffs, and 0.02 and 0.03 mg/kg for fish feedstuffs, respectively. This reliable, simple, and cost-effective method could be applied to the routine monitoring of TAP in animal feedstuffs.     

A Powerful Analytical Strategy Based on QuEChERS-Dispersive Solid-Phase Extraction Combined with Ultrahigh Pressure Liquid Chromatography for Evaluating the Effect of Elicitors on Biosynthesis of <Emphasis Type="Italic">trans</Emphasis>-Resveratrol in Grapes

A powerful methodological approach based on modified quick, easy, cheap effective, rugged, and safe (QuEChERS) extraction technique, followed by cleanup dispersive solid-phase extraction (dSPE) and combined with ultrahigh pressure liquid chromatography (UHPLC), is presented in this paper, for the rapid determination of the health-promoting phytoalexin, trans-resveratrol, in grapes. This is the first time, to our knowledge, that the combination QuEChERS-dSPE/UHPLC-PDA has been used for trans-resveratrol quantification in grapes. The method has been validated according to European Union Decision 2002/657/EC, in terms of selectivity, linearity, sensitivity, instrument LOD and method LOQ, accuracy, precision, extraction efficiency, and interference assessment. Validation experiments revealed sufficient linearity (R ²?=?0.9931) within the established concentration range confirmed by Mandel’s fitting test. The sensitivity was good with method detection limit (LOD) of 0.003 μg/mL and quantification limit (LOQ) of 0.010 μg/mL. Optimum QuEChERS-dSPE/UHPLC-PDA conditions led to recoveries of the target analyte in grape samples ranging between 75.1 and 99.7 % and precisions in the 0.9–4 % range (RSD, n?=?18). The method also afforded satisfactory results in terms of extraction efficiency and interference assessment and provides a valuable and promising tool for determination of trans-resveratrol in grapes with a high resolution within 8 min and a total analysis time of 11 min. The validated methodology was applied to evaluate the effect of the use of elicitors, namely benzothiadiazole and methyl jasmonate, in trans-resveratrol biosynthesis on Vitis vinifera Monastrell grapes. The results obtained revealed an increase of trans-resveratrol level of 2-fold for grapes treated with benzothiadiazole and 3.5-fold for grapes treated with methyl jasmonate.     

Determination of Ultraviolet Absorbers and Light Stabilizers in Food Packaging Bags by Magnetic Solid Phase Extraction Followed by High-Performance Liquid Chromatography

A sensitive and effective extraction technique based on magnetic solid phase extraction (MSPE) was developed for separating and detecting trace amounts of ultraviolet (UV) absorbers and light stabilizers in plastic packaging prior to high-performance liquid chromatography (HPLC). The magnetic Fe₃O₄/Au composites were synthesized by chemical co-precipitation, and a HAuCl₄ solution was added to the Fe₃O₄ NPs, followed by the addition of hydroxylamine hydrochloride as a reducing agent for Au shell formation. The main factors affecting the adsorbability of UV absorbers, such as the effects of pH of the sample solution, the amount of adsorbent, extraction time, and desorption conditions on the magnetic nanoparticles (MNPs), were investigated and optimized. The proposed method showed good linearity in the range from 0.75 to 100 μg/mL with good presenting regression coefficients (R ²) ≥0.9999. Low limits of detection (LODs) were achieved at 0.05, 0.14, and 0.04 μg mL^?1 for 2-hydroxy-4-n-octoxy-benzophenone (UV-531), 2-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl)-4-methylphenol (UV-326), and 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV-328), respectively. The mean recoveries were in the range from 88.06 to 100.29% at 2.5, 5, and 10 μg mL^?1 spiked levels, and the relative standard deviations (RSDs) were in the range from 0.98 to 3.56%.     

HPLC Determination of Spectinomycin in Feed Premixes and Dosage Forms Using 1-Naphthyl Isocyanate Precolumn Derivatization with UV Detection

A new, simple, and sensitive HPLC method was developed for the determination of spectinomycin hydrochloride in dosage forms and feed premixes through derivatization with 1-naphthyl isocyanate. The separation was achieved on a C₁₈ column using a mobile phase consisting of acetonitrile/water (50:50 v/v, pH 3.2) in a flow rate of 1 mL/min with UV detection at 230 nm. The factors influencing the derivatization reaction yields were carefully studied and optimized. The method was linear over the concentration range of 10–100 μg/mL with a limit of detection 0.25 μg/mL and limit of quantitation 1.75 μg/mL. The developed method was successfully applied to the analysis the drug in the commercial dosage forms and spiked feed premix samples; the average recoveries were 99.79 and 99.56, respectively. The analytical performance of the method was fully validated and the results were satisfactory. A proposal of the reaction pathway was presented.     

Rapid Screening and Determination of the Residues of Hormones and Sedatives in Milk Powder Using the UHPLC-MS/MS and SPE

A method based on the ultra-high-performance liquid chromatography tandem mass spectrometry for determination of the residues of sex hormones, glucocorticoids, and sedatives in milk powder was developed. The sample was extracted with the acetic acid-acetonitrile (1:99, v/v) twice, purified by the PRiME hydrophilic-lipophilic balance (HLB) cartridges and analyzed by the ultra-high-performance liquid chromatography-tandem mass spectrometry. The analytes were separated by the Waters Acquity UPLC? BEH C18 column (50 mm?×?2.1 mm, 1.7 μm) and determined using the electrospray ionization in the positive mode with the multiple reaction monitoring (MRM). The developed method was validated with the specificity, linearity and range, matrix effects, recovery, and precision. The results showed that the analytes were linear with the correlation of determinations (R²) higher than 0.991 in the corresponding ranges. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.1–1.1 μg kg^?1 and 0.3–3.8 μg kg^?1, respectively. The average recoveries of the analytes ranged from 78.5 to 107.0% with the relative standard deviations lower than 15%. The practical applicability was tested by analyzing real samples and the progesterone was observed in two samples.     

Simultaneous Determination of Seven Plant Growth Regulators in Melons and Fruits by Modified QuEChERS Coupled with Capillary Electrophoresis

A capillary electrophoresis method for the simultaneous determination of seven plant growth regulators including gibberellic acid, abscisic acid, 3-indole acetic acid, 3-indolepropionic acid, 3-indolebutyric acid, 2,4-dichlorophenoxyacetic acid, and 2-methyl-4-chlorophenoxyacetic acid in melons and fruits was established and validated. The samples were ultrasonically extracted with acidified acetonitrile and then dehydrated. The resulting solution was purified with modified QuEChERS method, and then dried-up under weak nitrogen flow, and the residue was redissolved with methanol-water (1:4, v/v) as sample solution. The electrophoretic separation was performed in an uncoated fused-silica capillary with borax buffer (pH 10.5) containing 29% of ethanol as the running buffer. The running voltage was 25 kV with the column temperature of 30 °C. The linear ranges of gibberellic acid, abscisic acid, and 2-methyl-4-chlorophenoxyacetic acid were from 0.10 to 8.0 μg/mL, while the others were from 0.05 to 4.0 μg/mL with the correlation coefficients greater than 0.997. The limits of detection and the limits of quantification of the method were in the range of 1.54–5.76 and 5.12–19.2 μg/kg, respectively. The recoveries of the method ranged from 69.0 to 109% with the relative standard deviations ranging from 2.01 to 15.4%. The proposed method has been successfully applied to the determination of plant growth regulator residues in 15 melon and fruit samples, and gibberellic acid and abscisic acid were detected in the samples.