Immunochemical approaches for detection of aflatoxin B1 in herbal medicines.

INTRODUCTION: Aflatoxin B1 (AFB1) is a toxic low-molecular-weight secondary metabolite of Aspergillus flavus and A. parasiticus. AFB1 was classified as a Group I carcinogen by the World Health Organisation for Research on Cancer in 1993. AFB1 is an unavoidable natural contaminant of some herbal medicine, able to cause serious health issues for humans consuming the related medicine. OBJECTIVE: Therefore, this study aimed to develop an efficient fluorescence polarisation immunoassay (FPIA) and a rapid, low-cost, and easy-to-use membrane-based flow-through immunoassay (MBA) for determination of AFB1 in herbal medicine Origanum vulgare L., Rubus idaeus L., Urtica dioica L. and Sorbus aucuparia L. RESULTS: A cut-off level of the developed MBA was 0.8 ppb. Validation of the developed test was performed with blank and spiked samples. Using three naturally contaminated or three artificially spiked samples. The FPIA showed a linear working range of 8.6 to 64 ppb, and a half maximal inhibitory concentration (IC50 ) of 24 ppb. CONCLUSION: The results were in good correlation with the enzymelinked immunosorbent assay (ELISA) results (the IC50 0.1 ppb). Both the sample preparation and analysis are simple, cost-effective and easy to perform on-site in non-laboratory environments. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used as a confirmatory technique.

Fluorescently labelled multiplex lateral flow immunoassay based on cadmium-free quantum dots.

A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures. They consisted of core/shell InP/ZnS QDs enrobed in a silica shell that allowed the water solubility (QD@SiO2). Then we applied the QD@SiO2 as novel, silica shell-encapsulated fluorescent labels in immunoassays for rapid multiplexed screening. Two mycotoxins, zearalenone and deoxynivalenol, were simultaneously detected in maize and wheat, since the two QD@SiO2 labelled conjugates emit at two different, individually detectable wavelengths. The cutoff values for the simultaneous determination were 50 and 500µgkg-1 for zearalenone and deoxynivalenol, respectively, in both maize and wheat. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the result.

Design of a sensitive fluorescent polarization immunoassay for rapid screening of milk for cephalexin.

In this paper we describe the development of a sensitive, fast, and easily performed fluorescence polarization immunoassay for determination of cephalexin in milk. The experimental work was performed to increase sensitivity and specificity. Therefore, the structures of the tracers were varied by synthesis of both cephalexin (CEX) and cephalotin (CET) conjugates with a variety of fluorescent labels. Two rabbit antisera containing antibodies against cephalexin and cephalotin were tested in homologous and heterologous combinations with the tracers. For every working antibody-tracer combination, the analytical conditions and cross-reactivity for structural analogues-cephalosporins and other antibiotics that could also be present in milk-were determined. It was found that the highest sensitivity was achieved by use of the homologous pair CET-EDF-anti-CET antibody (limit of detection (LOD) 0.4 µg kg(-1) for standard solutions prepared in buffer), but this combination was not appropriate because of high cross-reactivity with CET. For subsequent experiments, therefore, CEX- EDF-anti-CEX antibody were chosen (LOD 0.8 µg kg(-1) for standard solutions prepared in buffer). Part of this manuscript is devoted to the variation of precipitation agents for pretreatment of milk before analysis; milk is an extremely complicated matrix. The optimum protein precipitation agent was methanol. This technique for cephalexin determination was characterized by a limit of detection of 1 µg kg(-1). The method was validated by using naturally contaminated and spiked milk samples. The results obtained corresponded very well with those obtained by HPLC, which was used as confirmation method.

Hydrophilic, bright CuInS2 quantum dots as Cd-free fluorescent labels in quantitative immunoassay.

We report on the synthesis of core-shell CuInS2/ZnS quantum dots (QDs) in organic solution, their encapsulation with a PEG-containing amphiphilic polymer, and the application of the resulting water-soluble QDs as fluorescent label in quantitative immunoassay. By optimizing the methods for core synthesis and shell growth, CuInS2/ZnS QDs were obtained with a quantum yield of 50% on average after hydrophilization. After conjugation with an aflatoxin B1-protein derivative, the obtained QDs were used as fluorescent labels in microplate immunoassay for the quantitative determination of the mycotoxin aflatoxin B1. QDs-based immunoassay showed higher sensitivity compared to enzyme-based immunoassay.

Homogenous FRET-based fluorescent immunoassay for deoxynivalenol detection by controlling the distance of donor-acceptor couple.

Semiconductor quantum dots (QDs) are one of the most popular luminescent labels that are widely used in food and medical analysis. Their unique optical properties establish QDs as excellent tools for highly sensitive biosensors based on Förster resonance energy transfer (FRET). To provide a convenient analytical system with long-term optical stability, a FRET pair consisting of QDs as energy donor and gold nanoparticles (GNs) as energy acceptor was developed. Careful selection of donor and acceptor properties allowed to achieve a large Förster distance of 12.9 nm and to use full-size specific antibody. As the immunoreagents pair, mycotoxins were bound to proteins and then to GNs, while QDs were conjugated with specific antibodies. FRET was observed as a result of the immunocomplex formation. Contributions of FRET and inner filter effect on the quenching were evaluated separately. The quenching effect in the donor-acceptor pair was compared for proteins with different sizes. The developed homogeneous FRET-based immunoassay for the detection of deoxynivalenol (DON) is an example of a fast method for high-throughput control of mycotoxins. The quenching effect of FRET was observed with a limit of detection of 28 µg kg-1 of DON in spiked wheat samples.

Silanized Luminescent Quantum Dots for the Simultaneous Multicolor Lateral Flow Immunoassay of Two Mycotoxins.

A critical point for the successful development of a fluorescent quantum dot (QD)-based immunoassay is maintaining the high fluorescence quantum yield of QDs during hydrophilization and bioconjugation. In this paper, we carefully designed CdSe/CdS and CdSe/CdS/ZnS core-shell heterostructures and extended them with silica coating of different surface composition allowing preservation of fluorescence quantum yield as high as 70% in aqueous media. The silanized QDs containing epoxy and carboxy surface groups were bioconjugated with monoclonal antibodies. The synthesized fluorescent conjugates were used in a multicolor lateral flow immunoassay for simultaneous determination of two mycotoxins. Zearalenone and deoxynivalenol were chosen as a proof of concept. Cutoff levels for the zearalenone and deoxynivalenol detection were adjusted to be at 40 and 400 µg kg-1, respectively, complying with the European Commission regulation. Validation of the developed test was performed by analysis of 34 naturally contaminated maize and wheat samples; as a confirmatory method, LC-MS/MS was used.

Development of a validated direct injection-liquid chromatographic tandem mass spectrometric method under negative electrospray ionization for quantitation of nine microcystins and nodularin-R in lake water.

Microcystins (MCs) are cyclic heptapeptide toxins produced by various cyanobacterial genera that are toxic to both animals and humans. In this study, a novel strategy was proposed for the quantitation of nine MCs and Nodularin-R (NOD) in lake water using UHPLC-MS/MS under negative ionization mode, in which only centrifugation was employed during sample preparation. As a result, limits of quantification (LOQ) ranging from 0.05 to 0.1 µg/L for all studied compounds were obtained in water samples, which were lower than the results obtained using positive ionization mode. Additionally, validation was performed by spiking three different levels of MCs at 0.05 or 0.1, 0.5, 1.0 µg/L (n = 6). Recoveries ranged from 88.6% to 101.8%, and intraday and interday variability were lower than 12% and 14%, respectively, for all targeted compounds. Furthermore, the proposed method was applied to investigate microcystins contamination in fifty lake water samples collected in different regions in China. As a result, MC-LR, MC-RR, MC-YR, MC-WR, MC-LW, MC-LA, MC-LY, and MC-HilR were detected in lake water samples at trace level ranging from 0.06 to 0.37 µg/L. The obtained results indicated that it was necessary to monitor the presence of MCs in lake water, especially during regular cyanobacterial blooms during warmer months.

Soft glass multi-channel capillaries as a platform for bioimprinting.

Multi-channel capillaries (MC) formed from thousands individual microcapillaries with diameters ranging 10-100 µm are of a great interest for their use as platforms for molecular imprinting due to their relatively large surface area, high mechanical stability and possibility of facile integration in sensor systems. The manuscript proposes a new format of immunoassay based on imprinted protein immobilized on a MC inner surface modified with poly-l-lysine. The combination of the environmentally friendly, easy-to-produce and cheap recognition element with the carrier allowing to increase the assay sensitivity makes the described technique a perspective alternative for the existing screening tests. Two bioimprinting approaches were described. The imprinted protein (ovalbumin, OVA) primarily prepared separately and later immobilized on a MC structure was compared to the imprinted OVA directly prepared on the MC surface. Detection of a food contaminant zearalenone was chosen as a proof-of-concept. In a case of the immobilization of the primarily prepared imprinted OVA the reached limit of detection (LOD) was 0.8 ng/mL, and for the in-situ imprinted OVA the LOD was 0.12 ng/mL. The sensitivity of the developed bioimprinted assay was comparable to the commercially available ELISA kits for ZEN detection. The OVA in-situ imprinted on the MC surface was tested for the detection of ZEN in artificially spiked wheat samples. The high recovery values (88-112%) and good repeatability (RSD of 8.5-9.6%) were demonstrated allowing to conclude that the IPs-based MC-ELISA is a promising tool for analysis of the mycotoxin in complex matrices.

Antibody immobilization strategy for the development of a capacitive immunosensor detecting zearalenone.

A highly sensitive flow-injection capacitive immunosensor was developed for detection of the mycotoxin zearalenone (ZEN). Different strategies for immobilization of an anti-ZEN antibody on the surface of a gold electrode, i.e. polytyramine or self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (3-MPA) and lipoic acid (LA), were used and their performances were compared. The LA- and 3-MPA-based systems showed broad linear ranges for ZEN determination, i.e. from 0.010 nM to 10 nM and from 0.020 nM to 10 nM, respectively. Under optimal conditions, the LA-based immunosensor was capable of performing up till 13 regeneration-interaction cycles (with use of glycine HCl, pH 2.4) with a limit of detection (LOD) of 0.0060 nM, equivalent to 1.9 pg mL-1. It also demonstrated a good inter-assay precision (RSD < 10%). However, the tyramine-based capacitive immunosensor showed a bad repeatability (only 4 regeneration-interaction cycles were possible) and inter-assay precision (RSD > 15%) which did not allow sensitive and precise measurements. The LA-based method was compared with a direct ELISA. These results demonstrated that the label-free developed capacitive immunosensor had a better sensitivity and shorter analysis time in comparison with the direct microwell-plate format.

Silanization of quantum dots: Challenges and perspectives.

Here we report the facile strategies for synthesis of silica-coated quantum dots (QDs). Due to their small size, chemical and optical stability and high luminescence quantum yield these particles can be further applicable in chemical and biomedical analysis, and in vivo imaging. The article gives a structured and detailed overview of the silanization strategies and potential pitfalls and common technical issues in function of the stability of the obtaining QDs. Different types of QDs, In- and Cd-based, were compared in terms of their behaviour during the silica-encapsulation. To achieve that each silica nanoparticle contains only one single QD the silanization reaction was performed via a water-in-oil emulsion formation. We investigated a number of parameters which have an impact on the silica-coating process and on characteristics of the obtained nanoparticles, such as a nature of initial ligands on the QD surface, a silanization reagent, surfactant, ZnS shelling, etc. It was found that DDT is an optimal ligand for the silanization of InP-based quantum dots, but not for CdZnSeS-nanocomposites. GOPTES and CEST are the best silanization agents for covering InP-QDs, and the AOT/isooctane microemulsion is preferable for the silanization of CdZnSeS-composites.

Gel electrophoresis separation and origins of light emission in fluorophores prepared from citric acid and ethylenediamine.

We investigated light emission of hydrothermally treated citric acid and ethylenediamine (EDA) with various precursor ratios using gel-electrophoresis. We show that this relatively simple approach can deliver significant insights into the origins of photoluminescence. We found that products of the synthesis consist of both positively and negatively charged species and exhibit large dispersion in electrophoretic mobility (i.e. charge-to-size ratio). We observed that despite the large dispersion of the reaction products the blue light emission is confined to discrete bands clearly identifiable in the gel. We demonstrate clear evidence that this emission originates from the negatively charged light molecular fraction with the highest mobility which shows no excitation-dependent light emission. This molecular fluorophore exhibits spectral characteristics similar to previously reported 1,2,3,5-tetrahydro-5-oxo-imidazo[1,2-a]pyridine-7-carboxylic acid (IPCA). Secondary gel electrophoresis run performed on the bands extracted from the first run indicates that no further separation takes place. On the basis of our experimental results, we conclude that relatively stable binding exists between IPCA and EDA-derived product. Thus, the products of the reaction contain IPCA both in molecular form and in complexes with EDA-derived products. We conclude that excitation-dependent emission is related to the fluorophore binding to the positively charged EDA-derived products with a positive charge.

Capacitive sensor for detection of benzo(a)pyrene in water.

An affinity sensor based on capacitive transduction was developed to detect benzo(a)pyrene (BaP) in river water. Two types of recognition elements, the synthetic receptor analogues molecularly imprinted polymers (MIPs) and natural monoclonal antibody (mAb) were tested for this type of biosensor. Different polymerization strategies to obtain MIPs were compared. One approach comprised a preliminary batch synthesis of beads that were subsequently coupled covalently to an electrode surface. Another approach consisted of the in-situ synthesis of MIPs directly onto the electrode surface using electropolymerization. The latter approach provided the best results. To choose the optimal recognition element mAb and MIP-modified electrodes different sets were evaluated with regards to their sensitivity, selectivity, linear range and re-usability. The mAb-modified electrodes were considerably more sensitive toward BaP (ng L-1 range vs µg L-1 range for the MIP-modified one), and showed a broader linear working range than the MIP-modified electrodes. The latter revealed more suitable for group-selective determination of PAHs. The developed capacitive sensor was tested for the detection of BaP in naturally-contaminated water samples collected in different places of Ghent, Belgium. The results obtained with the sensor were coherent and reproducible, and were in a good agreement with the confirmation technique, HPLC-FlD.

Comparative study of colloidal gold and quantum dots as labels for multiplex screening tests for multi-mycotoxin detection.

Quantum dots (QDs) and colloidal gold nanoparticles (CG) were evaluated as labels for multiplex lateral flow immunoassay (LFIA) for determination of mycotoxins deoxynivalenol (DON), zearalenone (ZEN) and T2/HT2-toxin (T2/HT2) in cereal matrices. Both developed assays were based on the same immunoreagents (except for the labels), therefore their analytical characteristics could be objectively compared. For both LFIAs antigens (DON-ovalbumin (OVA), ZEN-OVA and T2-OVA) and rabbit anti-mouse immunoglobulin were immobilized on a nitrocellulose membrane as three test lines and one control line, respectively. Depending on the LFIA, monoclonal antibodies (mAb) against DON, ZEN and T2 were conjugated with CdSeS/ZnS QDs or CG. T2 and HT2 were detected by one test line (T2-OVA) with an anti-T2 mAb which showed 110% cross-reactivity with HT2. Both tests were developed in accordance with the legal limits and were developed in such a way that they had the same cut-off limits of 1000 µg kg-1, 80 µg kg-1 and 80 µg kg-1 for DON, ZEN and T2/HT2, respectively in order to allow a correct comparison. Applicability of these assays was demonstrated by analysis of naturally contaminated wheat samples. The results demonstrate that both the LFIAs can be used as rapid, cost-effective and convenient qualitative tool for on-site screening for simultaneous detection of DON, ZEN and HT2/T2 in wheat without special instrumentation. However, the QD-based LFIA consumed less immunoreagents and was more sensitive and economically beneficial. In addition, the results were easier to interpret, resulting in a lower false negative rate (<5%) which was in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes.

A Dual-Color Quantum Dots Encoded Frit-Based Immunoassay for Visual Detection of Aflatoxin M1 and Pirlimycin Residues in Milk.

Mycotoxins and antibacterial agents are the main chemical hazards that lead to several health problems. Nowadays, multiplex immunoassay is a primary goal throughout the world. Here, aflatoxin M1 and pirlimycin were selected as models, and a novel dual colorimetric encoded frit-based immunoassay was developed for simultaneously screening of aflatoxin M1 and pirlimycin residues in milk. This multiplex frit-based immunoassay combined two monoclonal antibodies to extend the spectrum of analytes and to enable detection of two classes of analytes in a single test. The cutoff values were 0.02 µg/kg for aflatoxin M1 and 0.5 µg/kg for pirlimycin, which satisfied the requirement to measure the maximum residue levels. The novel colorimetric frit-based immunoassay has the advantage of high throughput, short analysis time, reduced overall cost per assay, and can be used as a rapid screening technique for simultaneously detecting aflatoxin M1 and pirlimycin residues in milk.

Development of a Rainbow Lateral Flow Immunoassay for the Simultaneous Detection of Four Mycotoxins.

A multiplex lateral flow immunoassay (LFIA) for the determination of the mycotoxins deoxynivalenol, zearalenone, and T2/HT2-toxin in barley was developed with luminescent quantum dots (QDs) as label. The synthesized QDs were hydrophilized by two strategies, that is, coating with an amphiphilic polymer or silica. The water-soluble QDs were compared with regard to their bioconjugation with monoclonal antibody (mAb) and were tested on a LFIA. Silica-coated QDs that contained epoxy groups were most promising. Therefore, green, orange, and red epoxy-functionalized silica-coated QDs were conjugated with anti-ZEN, anti-DON, and anti-T2 mAb, respectively. The LFIA was developed in accordance with the European Commission legal limits with cutoff limits of 1000, 80, and 80 µg/kg for deoxynivalenol, zearalenone, and T2/HT2-toxin, respectively. The LFIA gave a fast result (15 min) with a low false-negative rate (<5%), and the results were easy to interpret without any sophisticated equipment.

Sensitive Flow-through Immunoassay for Rapid Multiplex Determination of Cereal-borne Mycotoxins in Feed and Feed Ingredients.

An easy-to-operate membrane-based flow-through test for multiplex screening of four mycotoxins (zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A) in a variety of cereal-based feed ingredients and compound feeds, such as wheat, barley, soybean, wheat bran, rice, rice bran, maize, rapeseed meal, and sunflower meal, and various types of complete feed (duckling feed, swine feed, broiler feed, piglet feed) was developed and validated. First, the antibodies were evaluated by enzyme-linked immunosorbent assay and then employed in the membrane rapid test. The cutoff levels for zearalenone, deoxynivalenol, aflatoxin B1, and ochratoxin A were 50, 200, 1, and 10 µg/kg, respectively, based on European regulations and consumers' requirements. As sample pretreatment, consecutive steps of extraction, dilution, solid-phase extraction by addition of C18 sorbent, and final filtration of supernatant were followed. Both the sample preparation and the analysis procedure were simple, cost-effective, and easy to perform on-site in a nonlaboratory environment. The impact of sample processing on the result of the experiment was investigated supported by experimental design. The validation procedure was performed on the basis of Commission Regulation 2006/401/EC. The numbers of false-positive and false-negative outcomes were <5%, going along with the Commission Decision 2002/657/EC. Liquid chromatography-tandem mass spectrometry was performed as a confirmatory technique.

Capacitive sensing of N-formylamphetamine based on immobilized molecular imprinted polymers.

A highly sensitive, capacitive biosensor was developed to monitor trace amounts of an amphetamine precursor in aqueous samples. The sensing element is a gold electrode with molecular imprinted polymers (MIPs) immobilized on its surface. A continuous-flow system with timed injections was used to simulate flowing waterways, such as sewers, springs, rivers, etc., ensuring wide applicability of the developed product. MIPs, implemented as a recognition element due to their stability under harsh environmental conditions, were synthesized using thermo- and UV-initiated polymerization techniques. The obtained particles were compared against commercially available MIPs according to specificity and selectivity metrics; commercial MIPs were characterized by quite broad cross-reactivity to other structurally related amphetamine-type stimulants. After the best batch of MIPs was chosen, different strategies for immobilizing them on the gold electrode's surface were evaluated, and their stability was also verified. The complete, developed system was validated through analysis of spiked samples. The limit of detection (LOD) for N-formyl amphetamine was determined to be 10µM in this capacitive biosensor system. The obtained results indicate future possible applications of this MIPs-based capacitive biosensor for environmental and forensic analysis. To the best of our knowledge there are no existing MIPs-based sensors toward amphetamine-type stimulants (ATS).

Simple, high efficiency detection of microcystins and nodularin-R in water by fluorescence polarization immunoassay.

This manuscript describes the development of a sensitive, fast and easily-performed fluorescence polarization immunoassay (FPIA) for detection of microcystins (MCs) and nodularin-R (NOD) in water. MCs and NOD, the most widespread cyanobacterial toxin are hepatotoxins and tumor promoters, and their acute exposure may result in severe health problems in animals and humans. The fluorescein-based tracers were synthesized, and for the first time preparative high performance liquid chromatography (HPLC) was employed for their purification. Optimal tracers for the analysis were selected by evaluating the immunochemical activity. Under the optimal conditions, the achieved limits of detection (LODs) for MC-LR and NOD were 0.86 and 0.95 µg L-1, respectively, providing a sufficient sensitivity to meet the provisional guideline value recommended by the World Health Organization (WHO). An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to confirm the accuracy and precision of the FPIA, and no obvious difference in recovery between these two methods was found. The correlation coefficients (R2) were higher than 0.968. The developed FPIA was easy-to-operate and could be completed within 10 min after simple filtration and adjustment of pH for water samples. The method can be easily extended for screening of other cyanotoxins, representing a versatile strategy for environmental sample analysis.

Synthesis, modification, bioconjugation of silica coated fluorescent quantum dots and their application for mycotoxin detection.

To create bright and stable fluorescent biolabels for immunoassay detection of mycotoxin deoxynivalenol in food and feed, CdSe/CdS/ZnS core-shell quantum dots (QDs) were encapsulated in silica nanoparticles through a water-in-oil reverse microemulsion process. The optical properties and stability of the obtained silica coated QDs (QD@SiO2), modified with amino, carboxyl and epoxy groups and stabilized with polyethylene glycol fragments, were characterized in order to assess their bioapplicability. The developed co-condensation techniques allowed maintaining 80% of the initial fluorescent properties and yielded stable fluorescent labels that could be easily activated and bioconjugated. Further, the modified QD@SiO2 were efficiently conjugated with antibodies and applied as a novel label in a microtiter plate based immunoassay and a quantitative column-based rapid immunotest for deoxynivalenol detection with IC50 of 473 and 20 ng/ml, respectively.

Sensitive QD@SiO2-based immunoassay for triplex determination of cereal-borne mycotoxins.

A sensitive tool for simultaneous quantitative determination of three analytes in one single well of a microtiter plate is shown for the first time. The developed technique is based on use of colloidal quantum dot enrobed into a silica shell (QD@SiO2) derivatives as a highly responsive label. Silica-coated quantum dots were prepared and subsequently modified via the co-hydrolysis with tetraethylorthosilicate (TEOS) and various organosilane reagents. Different surface modification schemes were compared in terms of applicability of the obtained particles for the multiplex immunoassay, e.g. stability and simplicity of their conjugation with biomolecules. As model system a multiplex immunosorbent assay for screening of three mycotoxins (deoxynivalenol, zearalenone and aflatoxin B1) in cereal-based products was realized via a co-immobilization of three different specific antibodies (anti- deoxynivalenol, anti-zearalenone and anti-aflatoxin B1) in one single well of a microtiter plate. Mycotoxins were simultaneously determined by labelling their conjugates with QD@SiO2 emitting in different parts of the visible spectrum. The limits of detection for the simultaneous determination were 6.1 and 5.3, 5.4 and 4.1, and 2.6 and 1.9µgkg(-1) for deoxynivalenol, zearalenone and aflatoxin B1 in maize and wheat, respectively. As confirmatory method, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used.