Ultrasensitive SERS aptasensor using Au@Ag bimetallic nanorod SERS tags for the selective detection of amantadine in foods.

Herein, a novel surface enhanced Raman spectroscopy (SERS) aptasensor was developed for amantadine (AMD) detection, based on magnetite nanoparticles coated with polyethylenimine, silver nanoclusters and aptamers (Fe3O4@PEI@AgNC-apt) as the capture probe and complementary DNA-modified gold nanorods (AuNRs@4-MPBA@Ag-c-DNA containing 4-mercaptophenylboric acid molecules) as the reporter probe. In the presence of AMD, the AMD and the reporter probe competed for the aptamer on the surface of the capture probe, resulting in the reporter probe detaching from the capture probe leading to a decrease in intensity of the SERS signal at 1067 cm-1 for 4-MPBA. Under optimal conditions, a good linear relationship was established between the SERS intensity at 1067 cm-1 and the logarithm of the AMD concentration over the range 10-6-102 mg L-1, with a LOD of 0.50 × 10-6 mg L-1. The AMD levels in spiked samples were evaluated using the SERS aptasensor, with good recoveries ranging from 90.57% to 113.49% being obtained.

Determination of Acetylamantadine by γ-Cyclodextrin-Assisted α-HL Nanopore for Potential Cancer Prediagnosis.

The high expression of Spermidine/spermine N1-acetyltransferase (SSAT-1) is an important indicator in early cancer diagnosis. Here, we developed a nanopore-based methodology with γ-cyclodextrin as an adaptor to detect and quantify acetylamantadine, the specific SSAT-1-catalyzed product from amantadine, to accordingly reflect the activity of SSAT-1. We employ γ-cyclodextrin and report that amantadine cannot cause any secondary signals in γ-cyclodextrin-assisted α-HL nanopore, while its acetylation product, acetylamantadine, does. This allows γ-cyclodextrin to practically detect acetylamantadine in the interference of excessive amantadine, superior to the previously reported ß-cyclodextrin. The quantification of acetylamantadine was not interfered with even a 50-fold amantadine and displayed no interference in artificial urine sample analysis, which indicates the good feasibility of this nanopore-based methodology in painless cancer prediagnosis. In addition, the discrimination mechanism is also explored by 2-D nuclear magnetic resonance (NMR) and nanopore experiments with a series of adamantane derivatives with different hydrophilic and hydrophobic groups. We found that both the hydrophobic region matching effect and hydrophilic interactions play a synergistic effect in forming a host-guest complex to further generate the characteristic signals, which may provide insights for the subsequent design and study of drug-cyclodextrin complexes.

[Determination of amantadine, rimantadine and dimethylamantadine residues in poultry by ultra-performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To establish a method for determination of amantadine, rimantadine and dimethylamantadine residues in poultry matrix by ultra-performance liquid chromatography-tandem mass spectrometry. METHODS: Poultry samples were extracted with acid acetonitrile, salting out, and then the organic phase was cleaned up by C_(18) and PSA. A Waters ACQUITYTM UPLC HSS T3 column(100 mm×2.1 mm, 1.7 mm)was used for liquid chromatography separation, ESI positive ion scan was used with multiple reaction monitoring(MRM) mode and quantified by matrix-matched external standard method. RESULTS: At the spiked level of 0.5, 1.0 and 5.0 µg/kg, the recoveries of each compound were in the range of 81.3%-91.1% with the relative standard deviations of 6.5%-11.3%. The qualitative limits of detections were 0.06-0.2 µg/kg and the quantitative limits were 0.2-0.5 µg/kg for the 3 target compounds. The established method was applied to the detection of the 3 target compounds in 30 poultry samples, and none of the target compounds exceeded the residue limits. CONCLUSION: The method is simple, rapid, high sensitivity and good stability, with a wide variety and a certain development. It can be used for the daily monitoring of the veterinary drug residues in poultry.

A novel method based on Ag-Au nanorings with tunable plasmonic properties for the sensitive detection of amantadine.

To prevent the toxic effect of amantadine (AMD) on humans, a sensitive and direct detection method is required. The conventional enzyme-linked immunosorbent assay (ELISA) usually shows a monochromatic gradient color variation with the concentration of the target; hence, it is not a sensitive method for naked-eye detection. In this work, Ag-Au nanorings with highly tunable plasmon properties were synthesized as colorimetric nanosensors. The growth of Ag on the hollow nanorings led to rich color variations. Ag-Au nanorings were integrated into ELISA for the sensitive detection of AMD with the naked eye. The proposed method showed high sensitivity for the qualitative and quantitative detection of AMD, the visible cut-off value (0.2 ng mL-1) and limit of detection (0.071 ng mL-1) were 10-fold and 4.7-fold lower, respectively, than those of conventional ELISA. This method showed a linear range of 0.08-2 ng mL-1 and 4-12 ng mL-1. The detection results of this method on 100 samples (food samples and municipal water) agreed well with those of liquid chromatography-tandem mass spectrometry. The proposed plasmonic ELISA has high sensitivity, easy operation, and naked-eye readout.

Dual-readout fluorescence quenching immunochromatographic test strips for highly sensitive simultaneous detection of chloramphenicol and amantadine based on gold nanoparticle-triggered photoluminescent nanoswitch control.

Herein, a novel fluorescence quenching immunochromatographic test strip (FQICTS) for simultaneous detection of chloramphenicol (CAP) and amantadine (AMD) was developed on the basis of inner filter effect (IFE), with the combination of gold nanoparticles (AuNPs) and highly luminescent green-emitting gold nanoclusters (AuNCs) as the IFE quencher/donor pair. The AuNPs could quench the excitation light and emission light of AuNCs and achieve a high IFE efficiency due to dual spectral overlapping. Under optimal conditions, the "turn-on" mode of the AuNCs-based dual-readout FQICTS showed good linearity for CAP detection in chicken samples from 0.05 ng/g to 10 ng/g, with a limit of detection (LOD) of 0.043 ng/g. The linear range of AMD is 0.5-50 ng/g, with LOD of 0.45 ng/g. The visual LODs of CAP and AMD in "turn-on" mode were 200 and 10 times lower than that in "turn-off" mode, respectively. The "turn-on" mode of FQICTS showed high recovery for detecting CAP (82.5-94.5%) and AMD (81.9-110.7%) spiked into chicken samples. The performance and practicability of the established method were verified with commercial enzyme-immunoassay kits, and good correlations were observed. Overall, the newly developed AuNCs-based dual-readout FQICTS is a promising on-site screening tool for rapid, high-sensitivity detection of multiple food contaminants in practical applications.

Comparison of two fluorescence quantitative immunochromatographic assays for the detection of amantadine in chicken muscle.

The two sensitive fluorescence quantitative immunochromatographic assays (FQICAs), background fluorescence quenching immunochromatographic assay (bFQICA) and time-resolved fluorescent immunochromatographic assay (TRFICA), play an important role increasingly in rapid detection technology for food safety. Amantadine (AMD), used extensively in virus infections in livestock and poultry, has been prohibited due to hazard concerns over public human health. Therefore, AMD was used as a model molecule in the FQICAs establishment and comparison based on the same bioreagents. The outstanding performance in technical parameters of the two FQICAs indicated that they could provide rapid, precise, reliable technical support for large-scale on-site screening for AMD detection. What's more, the systematic and comprehensive comparison of the two FQICAs would give useful suggestions for scientists and users in monitoring the harmful compounds.

Synthesis and characterization of tracers and development of a fluorescence polarization immunoassay for amantadine with high sensitivity in chicken.

Fluorescence polarization immunoassay (FPIA) is a homogeneous and rapid analytical method that is suitable for high-throughput screening of large numbers of samples. However, FPIA typically suffers from lower sensitivity than the well-established enzyme-linked immunosorbent assay (ELISA), limiting its wide application as an analytical tool that can be run with trace levels of an analyte. Herein, a highly sensitive FPIA for detecting amantadine (AMD) in chicken is described. To achieve high sensitivity, nine chemical tracers of AMD that employ different fluoresceins, fluorescein derivatives, and haptens were synthesized and paired with four previously produced monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of FPIA was investigated and discussed. We found that the tracers with a linear and shorter bridge between adamantane and fluorescein generally provided higher sensitivity. After optimization, N'-(1-adamantyl) ethylenediamine (AEDA), an AMD structural analogue labeled with fluorescein isothiocyanate (FITC), achieved the lowest IC50 value (1.0 ng/ml) in the FPIA, which was comparable to that of the heterologous ELISA format that used the same mAb7G2. We also investigated the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. The developed FPIA was applied to chicken to detect AMD residue, demonstrating a limit of detection (LOD) of 0.9 µg/kg with recoveries of 76.5-89.3% and coefficients of variation (CVs) below 14.5%. These results show that the proposed FPIA is an efficient, accurate, and convenient method for the rapid screening of AMD residues in chicken. PRACTICAL APPLICATION: The fluorescence polarization immunoassay (FPIA) was developed to determine and quantify amantadine (AMD) in chicken samples with high sensitivity. This homogeneous method avoids coating and washing steps and may provide high-throughput AMD screening in chicken in 10 min with high accuracy and precision. FPIA can be used as a monitoring tool and contribute significantly to the rapid detection of AMD in chicken.

Development of a molecularly imprinted microspheres-based microplate fluorescence method for detection of amantadine and rimantadine in chicken.

In this study, molecularly imprinted microspheres of a type capable of recognising amantadine and rimantadine were first synthesised, and three fluorescent tracers based on dansyl chloride, fluorescein isothiocyanate and 5-carboxytetramethylrhodamine were also synthesised. These reagents were used to develop and optimise a direct competitive fluorescence method on conventional 96-well microplate for detection of the two analytes. Results showed that this method achieved simple operation procedure, rapid assay process (30 min), high sensitivity (limits of detection 0.04-0.05 ng mL-1) and acceptable recycle performance (five times). After optimisation of several parameters, this method was used to detect amantadine and rimantadine in chicken muscle samples. Their recoveries from standards fortified blank samples were in the range of 62.3-93.7%. The analysis results for some real chicken samples were consistent with a confirmatory LC-MS/MS method. Therefore, this method could be used as a rapid, simple and accurate tool for routine screening the residues of amantadine and rimantadine in a large number of chicken muscle samples.

Simultaneous determination of five antiviral drug residues and stability studies in honey using a two-step fraction capture coupled to liquid chromatography tandem mass spectrometry.

An effective sample pretreatment method followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) was first developed for simultaneous determination of five antiviral drug residues including ribavirin, moroxydine, amantadine, rimantadine and memantine in honey. To adsorb analytes with different binding properties and overcome the interference of sugars and uridine as endogenous ribavirin structural analogs in honey, the target drugs were extracted with 1% formic acid and then purified by a phenylboronic acid (PBA) solid phase extraction cartridge using two-step fraction capture prior to LC-MS/MS analysis. This method was validated by analyzing honey samples from nine floral origins including miscellaneous flowers, citrus, vitex, rape, acacia, sunflower, linden, buckwheat and jujube spiking at multiple levels, and the recoveries ranged from 82.46% to 116.34%, with relative standard deviations (RSDs) less than 14.58%. The limits of detection (LODs) and limits of quantitation (LOQs) of moroxydine, ribavirin, amantadine, rimantadine, and memantine were 0.1-2 µg/kg and 0.2-5 µg/kg, respectively. Depletion experiments of five antiviral drugs in honey at different storage and process temperatures demonstrated that moroxydine can potentially be used as a drug to cure sacbrood disease in honeybees.

Homogeneous fluorescent immunoassay for the simultaneous detection of chloramphenicol and amantadine via the duplex FRET between carbon dots and WS2 nanosheets.

Herein, we proposed a duplex and homogeneous fluorescent immunoassay for the simultaneous detection of amantadine (AMD) and chloramphenicol (CAP) residue in chicken breast with both high sensitivity and short assay time. The immunoassay was based on the fluorescence resonance energy transfer (FRET) between hapten-labeled carbon dots (CDs) and antibody-modified WS2 nanosheets. To achieve the duplex FRET, polyethyleneimine-functionalized blue and green emissive CDs with separated emission were synthesized via a one-pot hydrothermal method and directly coupled with the haptens of AMD and CAP, serving as the energy donors. The antibodies were modified on the surface of WS2 nanosheets with high quenching efficiency to construct the energy acceptor. The specific immunoreaction could trigger the efficient FRET between the donors and the acceptors, causing the fluorescence quenching of CDs. The developed immunoassay was applied to simultaneously detect AMD and CAP, having the detection limit of 0.10 ng g-1 and 0.06 ng g-1, respectively.

Analysis of amantadine in Laminaria Japonica and seawater of Daqin Island by ultra high performance liquid chromatography with positive electrospray ionization tandem mass spectrometry.

A sensitive and validated method for determination of amantadine in Laminaria Japonica and seawater was established using ultra high performance liquid chromatography with positive electrospray ionization tandem spectrometry (UHPLC-ESI-MS/MS). Laminaria Japonica was extracted with acetonitrile containing formic acid (1%), then purified with 10.0 g anhydrous sodium sulfate, 0.50 g C18 and 0.50 g PSA powder. Seawater added 10.0 mL 0.20 mol/L hydrochloric acid was purified with MCX solid phase extraction (SPE) cartridge. After extraction and purification, the supernatant of Laminaria Japonica and eluate of seawater were evaporated to nearly dry under a gentle stream of nitrogen at 40 °C. Acetonitrile-0.1% formic acid in water (3/7, v/v) was adjusted to 1.00 mL final volume. An aliquot (10 µL) was injected into the C18 column for separation with the mobile phase of acetonitrile and 0.1% formic acid in water at 0.25 mL·min-1. Calibration curves were linear ranged from 1.00 ng/mL to 20.0 ng/mL. Mean recoveries were 73.5% to 95.8%, and limit of detection (LOD) and quantification (LOQ) were 0.50 µg/kg and 1.00 µg/kg for Laminaria Japonica. Mean recoveries were 75.8% to 93.4%, and LOD and LOQ were 0.50 ng/L and 1.00 ng/L for seawater. Based on the method above, Laminaria Japonica and seawater in Daqin Island were analyzed in February to June continuously, lgBAF3.71 (bioaccumulation factor), indicating a bioenrichment effect.

Fabrication and evaluation of a label-free piezoelectric immunosensor for sensitive and selective detection of amantadine in foods of animal origin.

A label-free piezoelectric immunosensor was fabricated and applied to the detection of the antiviral drug amantadine (AM) in foods of animal origin. Experimental parameters associated with the fabrication and measurement process were optimized and are discussed here in detail. The proposed piezoelectric sensor is based on an immunosuppression format and uses a portable quartz crystal microbalance (QCM) chip. It was found to provide a good response to AM, with a sensitivity and limit of detection (LOD) of 33.9 and 1.3 ng mL-1, respectively, as well as low cross-reactivity (CR, < 0.01%) with AM analogues. The immunosensor was further applied to quantify AM at three levels in spiked samples of typical foods of animal origin, and yielded recoveries of 83.2-93.4% and standard deviations (SDs, n = 3) of 2.4-4.5%, which are comparable to the results (recoveries: 82.6-94.3%; SDs: 1.7-4.2%) obtained using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method. Furthermore, the piezoelectric immunosensing chip can be regenerated multiple (at least 20) times with low signal attenuation (about 10%). A sample analysis can be completed within 50 min (sample pretreatment: about 40 min, QCM measurement: 5 min). These results demonstrate that the developed piezoelectric immunosensor provides a sensitive, accurate, portable, and low-cost analytical strategy for the antiviral drug AM in foods of animal origin, and this label-free detection method could also be applied to analyze other targets in the field of food safety. Graphical abstract.

Fluorescence immunoassay based on the inner-filter effect of carbon dots for highly sensitive amantadine detection in foodstuffs.

Immunoassays with ultra-high sensitivity for the rapid detection of chemical contaminants in food are urgently required. However, conventional enzyme-linked immunosorbent assay (ELISA) usually suffer from the moderate sensitivity. Herein, we aim to improve the sensitivity of conventional ELISA by employing the fluorescent carbon dots (CDs) as the signal probes based on the principle of inner filter effect (IFE). In this strategy, the enzymatically formed products of horseradish peroxidase/alkaline phosphatase efficiently quenched the CDs via the IFE. The absorption signal of the conventional ELISA was converted into the fluorescence signal. The fluorescent immunoassay was successfully developed and used to detect amantadine residues in chicken, achieving a limit of detection of 0.02 ng mL-1. The fluorescent immunoassay is a straightforward, extendable and general strategy and exhibits potential in detecting trace amounts of chemical contaminants in foodstuff.

Quantitative and rapid detection of amantadine and chloramphenicol based on various quantum dots with the same excitations.

Herein, we developed a sensitive and quantitative flow assay for simultaneous detection of amantadine (AMD) and chloramphenicol (CAP) in chicken samples based on different CdSe/ZnS quantum dots (QDs). In contrast to other reports, the QDs could be excited by the same excitations that lowered the requirements for the matching instruments. Under the optimal conditions, the strategy permitted sensitive detection of AMD and CAP in a linear range of 0.23 to 1.02 ng/g and 0.02 to 0.66 ng/g. The limits of detection were 0.18 ng/g and 0.016 ng/g, respectively. Moreover, the whole detection process could be completed within 20 min with no additional sophisticated instruments and complicated operations. Spiked samples were analyzed using both QD-based lateral flow immunoassay (QD-LFIA) and commercial ELISA kits with good correlation (R2 = 0.96). Moreover, this study laid the foundation and simplified the development of the requisite instrument. Graphical abstract ᅟ.

Discovery of potential transcriptional biomarkers in broiler chicken for detection of amantadine abuse based on RNA sequencing technology.

The aim of this study was to identify candidate transcriptional biomarkers so as to provide a new method for monitoring amantadine residues during the feeding of broiler chicken. RNA sequencing (RNA-seq) and bioinformatic analyses were conducted to examine the transcriptomic changes and screen differentially expressed genes (DEGs) in broiler chicken breast muscle and liver tissues treated with amantadine. The results indicated that a total of 170 DEGs were screened from broiler chicken breast muscle tissues after amantadine was fed. Among the genes, 120 were up-regulated and 50 were down-regulated. The gene ontology (GO) terms for these genes mainly existed in the areas of hydrolase activity, immune reaction and chemokine activity. The significantly enriched pathways in the Kyoto Encyclopedia for Genes and Genomes (KEGG) were in phagosomes, cell adhesion molecules (CAMs), lysosomes and extracellular matrix (ECM) receptors. From the broiler chicken liver tissues, 172 DEGs were screened, among which 116 were up-regulated and 56 were down-regulated. The GO terms of these DEGs were related to functions such as catalytic activities, metabolic activities, oxidation-reduction activities, immune reactions and cofactor binding. The significantly enriched KEGG pathways existed in metabolism, CAM, ECM receptor reaction and drug metabolism-cytochrome P450. According to the fold-change (FC), significance levels, functional annotations and possible biological processes of DEGs, 11 and 9 candidate DEGs related to amantadine treatment were further screened from broiler chicken breast muscle and liver tissues, respectively. In addition, the quantitative real-time polymerase chain reaction (qRT-PCR) verification showed exactly concordant results with the RNA-seq data. Principal components analysis (PCA) on the qRT-PCR data resulted in the separation of treated samples from the control samples in both tissues. The results provided a basis for identification of transcriptional biomarkers for detecting amantadine residues in broiler chicken breast muscle and liver tissues.

Simultaneous determination of antibiotics and amantadines in animal-derived feedstuffs by ultraperformance liquid chromatographic-tandem mass spectrometry.

In this study, a method for simultaneous determination of 40 antibiotics from 6 different drug families (7 quinolones, 5 penicillins, 8 macrolides, 2 lincosamides, 4 tetracyclines, and 13 sulfonamides) and 3 amantadines in animal-derived feedstuffs (ADF) through ultraperformance liquid chromatography-tandem mass spectrometry using a new lipid-removing solid-phase extraction column PRiME HLB was developed. The sample was extracted with 20 mL extraction solution and then purified and concentrated with PRiME HLB. The eluent was evaporated to dryness under nitrogen and analyzed with LC-MS/MS using acetonitrile and 0.1% formic acid as the mobile phase via gradient elution. Under the optimum conditions, recoveries were 65.8% to 104.4% with a relative standard deviation of <15% at spiked levels of 5.0 µg/kg to 100 µg/kg. The limits of detection ranged from 0.5 µg/kg to 5 µg/kg. 15 of the 43 drugs were found in ADF. Sulfonamides, fluoroquinolones, macrolides, tetracyclines, and amantadines were detected, with the highest levels reaching 325 µg/kg.

Magnetic-assisted biotinylated single-chain variable fragment antibody-based immunoassay for amantadine detection in chicken.

A sensitive competitive immunoassay with simple operation was developed for the detection of the anti-virus drug amantadine (AMD). The single-chain variable fragment (scFv) antibody against AMD was site-specific biotinylated and overexpressed as a secreted body in Escherichia coli AVB101. Horseradish peroxidase-labeled streptavidin-biotinylated scFv antibody (HRP-SA-BIO-scFv) could specifically bind to AMD-functionalized magnetic beads (MBs) and then the immune complexes were separated from the matrix solution by magnet. The concentration of the AMD could be known by the measurement of the signal produced by the horseradish peroxidase. The newly established assay provides a significant improvement in comparison to the conventional ELISA without SA-BIO signal amplification and MBs separation. The limit of detection and assay time was 0.64 vs. 8.4 ng/mL and 50 vs. 150 min, respectively. The recoveries ranged from 77.8 to 112% with the coefficient of variation less than 13%. The immunoassay exhibited an obvious cross-reactivity to rimantadine (84%), 1-(1-adamantyl)ethylamine (72%), and somantadine (63%). These results demonstrated that the developed immunoassay provided a sensitive, rapid, and accurate approach for the detection of AMD in chicken by employing MBs as solid phase and SA-BIO as signal amplification. When applied in natural chicken samples, the newly established method provided results consistent with those from UPLC-MS/MS, suggesting that the proposed method could be used for rapid screening of the target of interest; the new immunoassay could also be extended to other small molecular contaminants and thus represents a universal strategy for food safety analysis. Graphical abstract ᅟ.

Highly sensitive visual detection of amantadine residues in poultry at the ppb level: A colorimetric immunoassay based on a Fenton reaction and gold nanoparticles aggregation.

Colorimetric biosensors for the on-site visual detection of veterinary drug residues are required for food control in developing countries and other resource-constrained areas, where sophisticated instruments may not be available. In this study, we developed a highly sensitive immunoassay for amantadine residues in poultry. By introducing a novel signal generation strategy into an indirect competitive immunoassay, a highly sensitive assay for amantadine residues in chicken was achieved for naked eye readout at the part per billion (ppb) level. Signal amplification was achieved in the designed immunoassay by combining conventional indirect competitive enzyme-linked immunosorbent assay, Fenton reaction-regulated oxidation of cysteine, and gold nanoparticle aggregation. Therefore, the cascade reaction remarkably enhanced the assay sensitivity and led to a pronounced color change from red to dark purple in the solution, which could be easily distinguished with the naked eye even at approximately 1 µg kg-1 in poultry muscle. Moreover, the color change can be quantitatively assayed with a classic high-throughput plate reader for contaminated poultry samples. The limit of detection (LOD) was 0.51 nM (0.095 ng mL-1). The recovery rates for spiked chicken samples ranged from 78% to 84% with relative standard deviations <15%. Therefore, we propose that this immunoassay could be generally applicable for on-site detection in the field of food control.

Highly sensitive SERS immunosensor for the detection of amantadine in chicken based on flower-like gold nanoparticles and magnetic bead separation.

Here we report a novel ultrasensitive surface-enhanced Raman scattering (SERS) immunosensor based on the flower-like gold nanoparticles (AuNFs) and magnetic bead separation for homogeneous detection of amantadine (AMD) in chicken just by one-step. The 5, 5'-dithiobis (2-nitrobenzoicacid) (DTNB) modified AuNFs and N-(1-adamantyl) ethylenediamine (AEDA) conjugated denatured BSA (AEDA-dBSA) was used as the SERS nanoprobe. And the capture probe was anti-AMD monoclonal antibody (mAbs)-functionalized magnetic beads (MNBs-mAbs). An immunoreaction occurred between free AMD and SERS nanoprobe for competing limited binding sites of MNBs-mAbs. This work combined inherent sensitive property of SERS with antibody-antigen highly specificity recognition for the AMD detection. The analytical results showed that the SERS-based immunosensor was sensitive, simple and reliable with a limit of detection (LOD) of 0.005 ng/mL for AMD, which were 2 orders of magnitude better than an enzyme-linked immunosorbent assay based on the same immunoreagents. Analysis of AMD-spiked chicken samples revealed that the developed immunosensor provided accepted recoveries ranging from 74.76%-89.34% with coefficient of variation less than 15.04%. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants in food samples.

Simultaneous determination of amantadine and rimantadine in feed by liquid chromatography-Qtrap mass spectrometry with information-dependent acquisition.

A sensitive method for simultaneous determination of amantadine and rimantadine in feed was developed using an ultra-high-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC-Qtrap-MS) in the multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode, and employing the mixed cation exchange (MCX) solid-phase extraction column as sample cleanup and amantadine-d15 and rimantadine-d4 as internal standards, respectively. Compared to traditional MRM mode, for the targeted drugs in feed simultaneously both the secondary mass spectra and MRM information can be obtained using UHPLC-Qtrap-MS with MRM-IDA-EPI mode, and thus more accurate qualitative confirmation results achieved even at lower concentration of 0.2 µg/L in acceptable purity fit values. After optimization of sample preparation, good linearities (R > 0.9994) were obtained over the concentration range from 1 to 200 µg/L for amantadine and rimantadine. The precision was validated by intra-day and inter-day, and the relative standard deviations were all within 9.61%. Mean recoveries ranged from 76.1 to 112% at spiked concentrations of 0.5-100 µg/kg in three types of feed samples, including formula feed and complex concentrated feed for pigs and premix feed for chicken. The limits of detection (LODs) and quantification (LOQs) were 0.2 and 0.5 µg/kg for both drugs, respectively. The application in real feed samples further proved the accuracy and reliability of the developed method. This method provides an important tool to detect illegal uses of amantadine and rimantadine in feed. Graphical abstract Simultaneous quantitation and qualitative confirmation of amantadine and rimantadine in feed by MRM-IDA-EPI.