Development of a molecularly imprinted polymer-matrix solid-phase dispersion method for selective determination of β-estradiol as anabolic growth promoter in goat milk

A simple, fast, and sensitive method for determination of 17 β-estradiol (E2) in goat milk samples has been developed by combining selective molecularly imprinted matrix solid-phase dispersion (MIP–MSPD) and liquid chromatography with diode-array detection (DAD). The molecularly imprinted polymer was synthesized by use of 17β-estradiol as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker monomer, azobisisobutyronitrile as initiator, and acetonitrile as porogen, and was used as selective solid support for matrix solid-phase dispersion. The selected dispersant had high affinity for E2 in the goat milk matrix and the extract obtained was sufficiently clean for direct injection for HPLC analysis without any interferences from the matrix. The proposed MIP–MSPD method was validated for linearity, precision, accuracy, decision limit (CCα) and detection capability (CCβ), in accordance with European Commission Decision 2002/657/EC criteria. Linearity ranged from 0.3–10 μg g^?1 (correlation coefficient r ²?>?0.999). Mean recovery of E2 from goat milk samples at different spiked levels was between 89.5 and 92.2%, with RSD values within 1.3–2%. CCα and CCβ values were 0.36 and 0.39 μg g^?1, respectively. The developed MIP–MSPD method was successfully applied to direct determination of E2 in goat milk samples.

MALDI-TOF mass spectrometric determination of intact phospholipids as markers of illegal bovine milk adulteration of high-quality milk

In the dairy industry one of the most common frauds is mixing high-value milk (sheep’s and goats’) with milk of lower value (cows’). This illegal practice has commercial, ethical, and serious sanitary consequences because consumers can be exposed to hidden allergens contained in the undeclared cows’ milk. Here, we investigated the possibility of using matrix-assisted laser-desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as a rapid, sensitive, and accurate technique for detection of milk adulteration by analysis of phospholipid profiles. Lipid extracts of pure raw milk, commercial milk, and binary mixtures of cows’ and goats’ milk and cows’ and sheep’s milk (the concentrations of each milk varied from 0 % to 50 %) were analyzed with α-cyano-4-chlorocinnamic acid as matrix. The abundance ratio of the ions at m/z 703 and m/z 706 was found to be species-correlated and was used as marker of cows’ milk in sheep’s and goats’ milk. Furthermore, the procedure could potentially be applied to cheese samples, because peaks at m/z 703 and 706 were also found in several commercial cheese samples. This approach proved to be an efficient, rapid, and inexpensive method of detecting milk fraud.

Effect of mobile phase on resolution of the isomers and homologues of tocopherols on a triacontyl stationary phase

Reversed-phase liquid chromatographic (RPLC) separation of isomers and homologues of similar polarity is challenging. Tocopherol isomers and homologues are one such example. α, β, γ, and δ-tocopherols have been successfully separated by RPLC on triacontyl (C₃₀) stationary phase. System suitability was tested by using four mobile phases, and observed chromatographic separations of β and γ-tocopherols were compared. Comparison indicated that methanol–tert-butyl methyl ether (TBME) 95:5 (v/v) at a flow rate of 0.75 mL min^?1 was the best mobile phase. Detection systems were also evaluated on the basis of limit of quantification; it was concluded that fluorescence detection was best. The method was validated by analysis of two homologues and two isomers of tocopherol in sesame, maize, and soybean samples. MS coupled with an ESI interface in negative-ion mode [M ? H]^? was used for identification of individual components. It was concluded that addition of TBME to methanol was required to enhance the separation of β and γ-tocopherols, although methanol alone provided similar results. The applicability of the method to cereal, pulse, and oilseed samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the range 1.7–3.9 %. Recovery of tocopherols added to sesame samples ranged from 91 to 99 %.

A sensitive and specific ELISA for determining a residue marker of three quinoxaline antibiotics in swine liver

Methyl-3-quinoxaline-2-carboxylic acid (MQCA) is a possible residue marker for three quinoxaline veterinary medicines (olaquindox, mequindox, and quinocetone). The wide application of mequindox/quinocetone or the illegal use of olaquindox leads to MQCA residue in animal’s original food, thereby threatening the safety of human food. The indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) with a specific coating antigen and monoclonal antibody (MAB) was established and optimized for detecting MQCA in swine liver. Samples were acidified with 2 mol?l^?1 hydrochloric acid, extracted with ethyl acetate–hexane–isopropanol (8?+?1?+?1, v/v/v) and then detected by IC-ELISA. The logarithm correlation of standards to OD values ranged from 0.2 to 200 μg?l^?1, with IC₅₀ of 6.46 μg?l^?1. Negligible cross-reactivity happened to five quinoxaline antibiotics (olaquindox, mequindox, quinocetone, carbadox, and cyadox) and the metabolite of carbadox and cyadox (quinoxaline-2-carboxylic acid). When spiked with 1 to 100 μg?kg^?1 of MQCA, the recoveries ranged from 85.44 to 100.02 %, with the intra-assay coefficient of variation (CV) of 6.64–10.57 % and inter-assay CV of 7.29–10.88 %. The limit of detection for MQCA was 1.0 μg?kg^?1 in swine liver. Furthermore, incurred samples were detected by the IC-ELISA and then conformed by a reported LC/MS/MS method, it shown that there was good correlation between the two methods. All these results indicated that the IC-ELISA method is appropriate for surveillance MQCA residue in animal tissues.

Automated regenerable microarray-based immunoassay for rapid parallel quantification of mycotoxins in cereals

An automated flow-through multi-mycotoxin immunoassay using the stand-alone Munich Chip Reader 3 platform and reusable biochips was developed and evaluated. This technology combines a unique microarray, prepared by covalent immobilization of target analytes or derivatives on diamino-poly(ethylene glycol) functionalized glass slides, with a dedicated chemiluminescence readout by a CCD camera. In a first stage, we aimed for the parallel detection of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisins in cereal samples in a competitive indirect immunoassay format. The method combines sample extraction with methanol/water (80:20, v/v), extract filtration and dilution, and immunodetection using horseradish peroxidase-labeled anti-mouse IgG antibodies. The total analysis time, including extraction, extract dilution, measurement, and surface regeneration, was 19 min. The prepared microarray chip was reusable for at least 50 times. Oat extract revealed itself as a representative sample matrix for preparation of mycotoxin standards and determination of different types of cereals such as oat, wheat, rye, and maize polenta at relevant concentrations according to the European Commission regulation. The recovery rates of fortified samples in different matrices, with 55–80 and 58–79 %, were lower for the better water-soluble fumonisin B1 and deoxynivalenol and with 127–132 and 82–120 % higher for the more unpolar aflatoxins and ochratoxin A, respectively. Finally, the results of wheat samples which were naturally contaminated with deoxynivalenol were critically compared in an interlaboratory comparison with data obtained from microtiter plate ELISA, aokinmycontrol® method, and liquid chromatography–mass spectrometry and found to be in good agreement.

Gas chromatography–triple-quadrupole mass spectrometry for analysis of selected polyhalogenated pollutants in plants. Comparison of extraction methods

Different extraction methods, followed by gas chromatography coupled to triple quadrupole mass spectrometry, were evaluated for simultaneous extraction of seven polychlorinated biphenyls (PCBs) and six polybrominated diphenyl ethers (PBDEs) from common weeds. Pressurized liquid extraction (PLE) with in-cell clean-up, ultrasound-assisted extraction (UAE) with in-column clean-up, and UAE with dispersive solid-phase extraction (dSPE) clean-up were evaluated and compared. In-cell clean-up with 4 g Florisil and 0.5 g graphitized carbon black (GCB) and two extraction cycles of 10 min with n-hexane–ethyl acetate 80:20 (v/v) at 60 °C were used for the PLE procedure. UAE with in-column clean-up was conducted under conditions similar to those reported for the PLE method whereas in UAE with dSPE clean-up purification of the extract was performed after extraction using primary and secondary amine sorbent (PSA) and GCB. Recovery from 82 to 104 % was obtained for all the compounds by PLE whereas, in general, lower extraction efficiency was obtained by UAE with in-column clean-up (especially for BDE-17 and BDE-183, for which recovery was 70 and 41 %, respectively) and by UAE with dSPE clean-up, for which the main drawback is that BDE-183 cannot be extracted. Finally, PLE was used for analysis of PCBs and PBDEs in different plants (Lolium rigidum, Lactuca serriola, Malva sylvestris, and Verbascum thapsus) collected from residential and/or rural areas of Madrid (Spain). Several of the analyzed compounds were detected at low levels in these plants, but only PCB-153 could be quantified.

Fluorescent probe for copper(II) ion based on a rhodamine spirolactame derivative, and its application to fluorescent imaging in living cells

A fluorescent probe for Cu(II) ion is presented. It is based on the rhodamine fluorophore and exhibits high selectivity and sensitivity for Cu(II) ion in aqueous methanol (2:8, v/v) at pH 7.0. The response is based on a ring opening reaction and formation of a strongly fluorescent 1:1 complex. The response is reversible and linear in the range between 50?nM and 900?nM, with a detection limit of 7.0?nM. The probe was successfully applied to fluorescent imaging of Cu(II) ions in HeLa cells.

Development of monolith-based stir bar sorptive extraction and liquid chromatography tandem mass spectrometry method for sensitive determination of ten sulfonamides in pork and chicken samples

A highly sensitive method was developed for the simultaneous determination of ten sulfonamides in pork and chicken samples by monolith-based stir bar sorptive extraction (SBSE) coupled to high-performance liquid chromatography tandem mass spectrometry. The samples were freeze-dried and extracted by acetonitrile, then enriched and further extracted by SBSE which was based on poly(vinylphthalimide-co-N,N-methylenebisacrylamide) monolith (SBSE-VPMB) as coating. To achieve optimum extraction performance of SBSE for sulfonamides, several parameters, including pH value and ionic strength in the sample matrix and extraction and desorption time, were investigated in detail. Under the optimal conditions, the limits of detection (S/N?=?3) for target sulfonamides were 1.2–6.1 ng/kg in pork and 2.0–14.6 ng/kg in chicken, respectively. Real samples spiked at the concentration of 0.5 and 5.0 μg/kg showed recoveries above 55 % and relative standard deviations below 12 %. At the same time, the extraction performances of target sulfonamides on SBSE-VPMB were compared with other SBSE based on porous monolith and commercial SBSE.

Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography–tandem mass spectrometry

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography–tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 μmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk

To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab μL^?1 milk and 0.4 ng mL^?1 Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n?=?8) or non-transgenic (n?=?7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits.

Milk and serum standard reference materials for monitoring organic contaminants in human samples

Four new Standard Reference Materials (SRMs) have been developed to assist in the quality assurance of chemical contaminant measurements required for human biomonitoring studies, SRM 1953 Organic Contaminants in Non-Fortified Human Milk, SRM 1954 Organic Contaminants in Fortified Human Milk, SRM 1957 Organic Contaminants in Non-Fortified Human Serum, and SRM 1958 Organic Contaminants in Fortified Human Serum. These materials were developed as part of a collaboration between the National Institute of Standards and Technology (NIST) and the Centers for Disease Control and Prevention (CDC) with both agencies contributing data used in the certification of mass fraction values for a wide range of organic contaminants including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, polybrominated diphenyl ether (PBDE) congeners, and polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners. The certified mass fractions of the organic contaminants in unfortified samples, SRM 1953 and SRM 1957, ranged from 12 ng/kg to 2200 ng/kg with the exception of 4,4′-DDE in SRM 1953 at 7400 ng/kg with expanded uncertainties generally <14 %. This agreement suggests that there were no significant biases existing among the multiple methods used for analysis.

Dual cloud point extraction coupled with hydrodynamic-electrokinetic two-step injection followed by micellar electrokinetic chromatography for simultaneous determination of trace phenolic estrogens in water samples

A dual cloud point extraction (dCPE) off-line enrichment procedure coupled with a hydrodynamic–electrokinetic two-step injection online enrichment technique was successfully developed for simultaneous preconcentration of trace phenolic estrogens (hexestrol, dienestrol, and diethylstilbestrol) in water samples followed by micellar electrokinetic chromatography (MEKC) analysis. Several parameters affecting the extraction and online injection conditions were optimized. Under optimal dCPE–two-step injection–MEKC conditions, detection limits of 7.9–8.9 ng/mL and good linearity in the range from 0.05 to 5 μg/mL with correlation coefficients R ²?≥?0.9990 were achieved. Satisfactory recoveries ranging from 83 to 108 % were obtained with lake and tap water spiked at 0.1 and 0.5 μg/mL, respectively, with relative standard deviations (n?=?6) of 1.3–3.1 %. This method was demonstrated to be convenient, rapid, cost-effective, and environmentally benign, and could be used as an alternative to existing methods for analyzing trace residues of phenolic estrogens in water samples.

Miniaturised enzymatic conductometric biosensor with Nafion membrane for the direct determination of formaldehyde in water samples

A new conductometric enzyme-based biosensor was developed for the determination of formaldehyde (FA) in aqueous solutions. The biosensor was prepared by cross-linking formaldehyde dehydrogenase from Pseudomonas putida with bovine serum albumin in saturated glutaraldehyde vapours (GA) at the surface of interdigitated gold microelectrodes. Nicotinamide adenine dinucleotide cofactor (NAD⁺) was added in solution at each measurement to maintain enzyme activity. Addition of a Nafion layer over the enzyme modified electrode resulted in a significant increase of biosensor signal due to enhanced accumulation of protons generated by enzymatic reaction at the electrode surface. Different parameters affecting enzyme activity or playing a role in ionic transfer through the Nafion membrane were optimised. In optimal conditions (0.045 mg enzyme, 30 min exposure to GA, 0.3 μL of a 1 % (v/v) Nafion solution deposit, measurement in 5 mM phosphate buffer pH 7 containing 20 μM NAD⁺), the biosensor signal was linear up to 10 mM FA, and the detection limit was 18 μM. Relative standard deviations calculated from five consecutive replicates of FA solutions were lower than 5 % in the 1–10 mM range. The biosensor was successfully applied to the determination of FA in spiked water samples (tap water and Rhone river water), with recoveries in the 95–110 % range.

A quantitative and comprehensive method to analyze human milk oligosaccharide structures in the urine and feces of infants

Human milk oligosaccharides (HMOs), though non-nutritive to the infant, shape the intestinal microbiota and protect against pathogens during early growth and development. Infant formulas with added galacto-oligosaccharides have been developed to mimic the beneficial effects of HMOs. Premature infants have an immature immune system and a leaky gut and are thus highly susceptible to opportunistic infections. A method employing nanoflow liquid chromatography time-of-flight mass spectrometry (MS) is presented to simultaneously identify and quantify HMOs in the feces and urine of infants, of which 75 HMOs have previously been fully structurally elucidated. Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS was employed for high-resolution and rapid compositional profiling. To demonstrate this novel method, samples from mother–infant dyads as well as samples from infants receiving infant formula fortified with dietary galacto-oligosaccharides or probiotic bifidobacteria were analyzed. Ingested oligosaccharides are demonstrated in high abundance in the infant feces and urine. While the method was developed to examine specimens from preterm infants, it is of general utility and can be used to monitor oligosaccharide consumption and utilization in term infants, children, and adults. This method may therefore provide diagnostic and therapeutic opportunities.

Sensitive routine liquid chromatography–tandem mass spectrometry method for serum estradiol and estrone without derivatization

The need for a routinely applicable assay to measure low estradiol levels in adult men, postmenopausal women, and young adolescents was recently discussed in an Endocrine Society position statement. Our aim was to develop a sensitive liquid chromatography–tandem mass spectrometry method for estradiol and estrone in human serum without the need for derivatization or extended extraction protocols. After protein precipitation of serum with a mixture of methanol/acetonitrile (85/15) (v/v) containing isotopic internal standards (17β-estradiol-16,16,17-d ₃ and estrone-2,3,4-¹³C), we quantified estradiol and estrone by two-dimensional liquid chromatography–tandem mass spectrometry with electrospray ionization in the negative mode monitoring 5?×?271.20→145.00 (17β-estradiol) and 269.20→145.00 (estrone). Sensitivity was increased by using fluoride and summation of 5 identical transitions for estradiol. Our method was analytically validated, compared against direct immunoassays using serum of 25 adult men, and clinically tested by measuring samples of 3 men at baseline and after chemical castration, 30 postmenopausal women and 15 patients receiving aromatase inhibitors. Total imprecision was below 20 % for the low quality controls. Limit of quantification was 1.3 ng/L (4.8 pmol/L) for estradiol and 1.2 ng/L (4.4 pmol/L) for estrone. Estradiol in Certified Reference Material BCR-576 was within specified uncertainty limits. No significant ion suppression or interference was observed. Our method showed modest correlation with direct immunoassay for estradiol (r ²?=?0.64) but no correlation for estrone (r ²?=?0.12). Patient sample results were within expected ranges. In conclusion, we developed a routinely applicable liquid chromatography–tandem mass spectrometry method for estradiol and estrone measurement which is sensitive enough for use in men, postmenopausal women, and young adolescents.

Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay

A fast and robust high-throughput ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC–TOF MS) profiling method was developed and successfully applied to discriminate a total of 78 Bacillus cereus strains into no/low, medium and high producers of the emetic toxin cereulide. The data obtained by UPLC–TOF MS profiling were confirmed by absolute quantitation of cereulide in selected samples by means of high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) and stable isotope dilution assay (SIDA). Interestingly, the B. cereus strains isolated from four vomit samples and five faeces samples from patients showing symptoms of intoxication were among the group of medium or high producers. Comparison of HEp-2 bioassay data with those determined by means of mass spectrometry showed differences, most likely because the HEp-2 bioassay is based on the toxic action of cereulide towards mitochondria of eukaryotic cells rather than on a direct measurement of the toxin. In conclusion, the UPLC–electrospray ionization (ESI)–TOF MS and the HPLC–ESI–MS/MS–SIDA analyses seem to be promising tools for the robust high-throughput analysis of cereulide in B. cereus cultures, foods and other biological samples.

A multiresidual method based on ion-exchange chromatography with conductivity detection for the determination of biogenic amines in food and beverages

In the present work a sensitive and accurate method by ion chromatography and conductimetric detection has been developed for the determination of biogenic amines in food samples at microgram per kilogram levels. The optimized extraction procedure of trimethylamine, triethylamine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine from real samples, as well as the separation conditions based on a multilinear gradient elution with methanesulfonic acid and the use of a weak ionic exchange column, have provided excellent results in terms of resolution and separation efficiency. Extended calibration curves (up to 200 mg/kg, r?>?0.9995) were obtained for all the analyzed compounds. The method gave detection limits in the range 23–65 μg/kg and quantification limits in spiked blank real samples in the range 65–198 μg/kg. Recovery values ranged from 82 to 103 %, and for all amines, a good repeatability was obtained with precision levels in the range 0.03–0.32 % (n?=?4). The feasibility and potential of the method were tested by the analysis of real samples, such as tinned tuna fish, anchovies, cheese, wine, olives, and salami.

Occurrence of venlafaxine residues and its metabolites in marine mussels at trace levels: development of analytical method and a monitoring program

Coastal areas are subject to growing pressures and impacts because of the increase in human activities. Lipophilic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs) or polychlorinated biphenyls (PCBs), have been monitored for decades within monitoring programs. However, until now, little information on the detection of so-called “emerging contaminants” such as hydrophilic organic compounds in the marine environment and no data on its metabolites or transformation products in marine organisms is available. In this report, a sensitive analytical methodology for identification and confirmation of venlafaxine (VEN) residues and five of its main metabolites in the marine mussels Mytilus galloprovincialis was validated. The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was developed to quantify these compounds at trace levels by liquid chromatography coupled to high-resolution mass spectrometry. The method was then applied to marine mussels collected from the Mediterranean Sea in southeastern France. Residues of the antidepressant VEN were occasionally detected at ng/g dw level. In addition, the approach allowed us to identify several transformation products in the analyzed samples. N-desmethylvenlafaxine (NDV) was the most frequently detected metabolite followed by N,O-di-desmethylvenlafaxine (NODDV).

Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk

A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.