Immuno-Enriched Microspheres - Magnetic Blade Spray-Tandem Mass Spectrometry for Domoic Acid in Mussels.

Paramagnetic microspheres can be used in planar array fluorescence immunoassays for single or multiplex screening of food contaminants. However, no confirmation of the molecular identity is obtained. Coated blade spray (CBS) is a direct ionization mass spectrometry (MS) technique, and when combined with triple quadrupole MS/MS, it allows for rapid confirmation of food contaminants. The lack of chromatography in CBS, though, compromises the specificity of the measurement for unequivocal identification of contaminants, based on the European Union (EU) regulation. Therefore, a rapid and easy-to-use immuno-magnetic blade spray (iMBS) method was developed in which immuno-enriched paramagnetic microspheres replace the coating of CBS. The iMBS-MS/MS method was fully optimized, validated in-house following the EU 2021/808 regulation, and benchmarked against a commercial lateral flow immunoassay (LFIA) for on-site screening of DA. The applicability of iMBS-MS/MS was further demonstrated by analyzing incurred mussel samples. The combination of immunorecognition and MS/MS detection in iMBS-MS/MS enhances the measurement's selectivity, which is demonstrated by the rapid differentiation between the marine toxin domoic acid (DA) and its structural analog kainic acid (KA), which cannot be achieved with the LFIA alone. Interestingly, this first-ever reported iMBS-MS/MS method is generic and can be adapted to include any other immuno-captured food contaminant, provided that monoclonal antibodies are available, thus offering a complementary confirmatory analysis approach to multiplex immunoassay screening methods. Moreover, thanks to its speed of analysis, iMBS-MS/MS can bridge the logistics gap between future large-scale on-site testings using LFIAs and classical time-consuming confirmatory MS analysis performed in official control laboratories.

From Smartphone Lateral Flow Immunoassay Screening to Direct MS Analysis: Development and Validation of a Semi-Quantitative Direct Analysis in Real-Time Mass Spectrometric (DART-MS) Approach to the Analysis of Deoxynivalenol.

In current food safety monitoring, lateral flow immunoassays (LFIAs) are widely used for rapid food contaminant screening. Recent advances include smartphone readouts, offering semi-quantitative analysis of LFIAs with time, location, and data transfer in case of on-site testing. Following the screening, the next step in the EU regulations is confirmation by, e.g., liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this work, using direct analysis in real time ambient ionization and triple quadrupole MS/MS (DART-QqQ-MS/MS), we achieved rapid confirmation of the identity of the substance(s) causing the LFIA result. In the workflow proposed, an individual performs the (on-site) smartphone LFIA screening, and when the result is suspect, an identification LFIA (ID-LFIA) strip is developed with the same sample extract. The ID-LFIA can be dissociated and rapidly analyzed in a control laboratory with DART-QqQ-MS/MS. The ID-LFIA consists of multiple lines of monoclonal antibodies against the mycotoxin deoxynivalenol, acting as a bioaffinity trap. The ID-LFIA/DART-QqQ-MS/MS approach has been developed and validated, along with the screening smartphone LFIA, and has demonstrated its applicability by analyzing incurred and spiked samples. The developed approach has been critically compared with our previous direct electrospray ionization MS method and was found to provide highly complementary information on the total deoxynivalenol contamination in the sample.

Direct analysis of lateral flow immunoassays for deoxynivalenol using electrospray ionization mass spectrometry.

Lateral flow immunoassays (LFIAs) are widely used for rapid food safety screening analysis. Thanks to simplified protocols and smartphone readouts, LFIAs are expected to be increasingly used on-site, even by non-experts. As a typical follow-up in EU regulatory settings, suspect samples are sent to laboratories for confirmatory analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, re-analysis by LC-MS/MS is laborious and time-consuming. In this work, an identification LFIA (ID-LFIA) approach followed by quadrupole-orbitrap MS or triple quadrupole MS/MS analysis is presented. As a proof of concept, a dedicated ID-LFIA strip was developed for the mycotoxin deoxynivalenol (DON) following its initial screening by a commercial smartphone LFIA. The ID-LFIA strip can be simply immersed in the same sample extract used for the smartphone LFIA screening, and next, DON is retrieved from the monoclonal antibody with a dissociation solution consisting of methanol/ammonia. The solution thus obtained was analyzed directly in MS in order to rapidly confirm the presence of DON and any cross-reacting species. The protocol developed is capable of coping with severe ion suppression caused by LFIA buffers and nitrocellulose substrate residues. Initial analysis of blank, spiked, and incurred samples showed that the newly developed ID-LFIA-MS method was able to confirm the presence or absence of mycotoxins in the samples previously analyzed by LFIA and also differentiate between DON and DON 3-glucoside yielding the positive screening result. The concept and technique developed are envisaged to complement on-site screening and confirmation of any low molecular weight contaminant in future food control frameworks. Graphical abstract.

Cytosine-rich mismatched DNA aptamer combined with superparamagnetic photonic crystal sensing material for the specific visual detection of silver ions.

DNA aptamer superparamagnetic photonic crystals (DSPCs), enriched with a highly selective cytosine-rich mismatched single-stranded DNA aptamer (CRDA), were successfully employed in a novel visual detection strategy for the detection of silver ions (Ag+). The technologies of superparamagnetic colloidal nanospheres (SCNs), DNA aptamer, and photonic crystals were combined to fabricate DPSCs. The aptamer was immobilized via electrostatic adsorption with amino groups that were chemically introduced on the surface of the SCNs, forming D-NH-SCNs. The detection is achieved by forming an Ag+ complex (C-Ag+-C) between Ag+ and D-NH-SCN. The DSPCs assembled under a magnetic field by D-NH-SCNs effectively detected Ag+ in the range of 1 µg/L to 5 mg/L, corresponding to the critical concentration range for heavy metals in drinking water. During the detection, the DSPC exhibited a wavelength blueshift from 652.8 nm to 626.4 nm (26.4 nm), as well as changes in reflection intensity. Notably, when detecting Ag+, a change in DSPC color from orange to yellow was observed. In summary, the developed visual detection material facilitates direct Ag + sensing. In the future, different DNA aptamers will be modified further to detect various targets in the fields of medicine, environmental monitoring, and food safety.

Immunoaffinity Plastic Blade Spray Mass Spectrometry for Rapid Confirmatory Analysis of Food Contaminants.

The lack of chromatographic separation in ambient and direct mass spectrometry (MS) ionization techniques jeopardizes the overall selectivity of the developed methods. Incorporating a biosensing element at the ionization source could compensate for that inherent lack of selectivity. Thus, a simplified immunoaffinity-direct MS technique was developed, immunoaffinity blade spray (iBS), featuring a conductive polystyrene blade material. In iBS, the generic coating used in conventional coated blade spray is replaced with a layer of highly specific monoclonal antibodies (mAbs), while the stainless steel is replaced with conductive polystyrene to allow for simple ELISA platelike hydrophobic immobilization of mAbs. Because of its high relevance for climate change-induced food safety issues, the mycotoxin deoxynivalenol (DON) was chosen as a model substance. Following a rapid extraction from wheat flour, DON is immuno-captured, and the blade is positioned in front of the MS for direct iBS-MS/MS analysis. The method's applicability was demonstrated by analyzing spiked and incurred wheat flour samples, omitting the need for time-consuming chromatographic separation. Apart from DON, cross-reacting DON conjugates could be successfully analyzed as well. The direct iBS-MS/MS method is generic and adaptable to detecting any analyte in sample extracts, provided that specific mAbs are available.

ASSURED Point-of-Need Food Safety Screening: A Critical Assessment of Portable Food Analyzers.

Standard methods for chemical food safety testing in official laboratories rely largely on liquid or gas chromatography coupled with mass spectrometry. Although these methods are considered the gold standard for quantitative confirmatory analysis, they require sampling, transferring the samples to a central laboratory to be tested by highly trained personnel, and the use of expensive equipment. Therefore, there is an increasing demand for portable and handheld devices to provide rapid, efficient, and on-site screening of food contaminants. Recent technological advancements in the field include smartphone-based, microfluidic chip-based, and paper-based devices integrated with electrochemical and optical biosensing platforms. Furthermore, the potential application of portable mass spectrometers in food testing might bring the confirmatory analysis from the laboratory to the field in the future. Although such systems open new promising possibilities for portable food testing, few of these devices are commercially available. To understand why barriers remain, portable food analyzers reported in the literature over the last ten years were reviewed. To this end, the analytical performance of these devices and the extent they match the World Health Organization benchmark for diagnostic tests, i.e., the Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end-users (ASSURED) criteria, was evaluated critically. A five-star scoring system was used to assess their potential to be implemented as food safety testing systems. The main findings highlight the need for concentrated efforts towards combining the best features of different technologies, to bridge technological gaps and meet commercialization requirements.

Development and validation of a hydrophilic interaction liquid chromatography method for the quantitation of impurities in fixed-dose combination tablets containing rosuvastatin and metformin.

A hydrophilic interaction liquid chromatography method with diode array detection (HILIC-DAD) was developed and validated for the simultaneous determination of impurities in extended-release fixed-dose combination tablets containing rosuvastatin and metformin in a ratio 1:100. The analytes were separated by hydrophilic interaction liquid chromatography using an XBridge®-HILIC analytical column under isocratic elution. The mobile phase was composed of ammonium formate at 150 mM containing 0.05% diethylamine (pH 8.5)/acetonitrile, 4/96 (v/v) and pumped at a flow rate of 0.5 mL min-1. Method validation was performed according to ICH guidelines. The calibration curves for rosuvastatin, metformin and their seven impurities showed good linearity (r > 0.994) within the calibration ranges tested. The intra- and inter-day R.S.D. values were less than 4.5%, while the relative percentage error Er was less than 2.7% for all compounds. Accelerated stability studies performed under various stress conditions including hydrolysis, oxidation and heat proved the selectivity of the procedure. A run time of less than 25 min for each sample made it possible to analyze a large number of samples per day. The method is the first reported application of HILIC for the analysis of impurities in fixed-dose combination tablets containing rosuvastatin and metformin and it can be used for the quality control of these drugs.

A porous graphitized carbon LC-ESI/MS method for the quantitation of metronidazole and fluconazole in breast milk and human plasma.

Information on drug transfer into the breast milk is essential to protect the infant from undesirable adverse effects of maternal consumption of drugs and to allow effective pharmacological treatment of breastfeeding mothers. Metronidazole and fluconazole are two drugs frequently used in nursing women to treat various infections, thus questioning infant's safety due to drug exposure through breast milk. In this article a porous graphitized carbon LC/ESI-MS assay was developed for the quantitation of metronidazole and fluconazole in breast milk and human plasma. The assay was based on the use of 150 µL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the LC/ESI-MS system. All analytes and the internal standard, ropinirole, were separated by using a porous graphitized carbon analytical column (150 × 2.1 mm i.d., particle size 5 µm) with isocratic elution. The mobile phase consists of 55% acetonitrile in water acidified with 0.1% concentrated formic acid and pumped at a flow rate of 0.25 mL min-1. The assay was linear over a concentration range of 0.1 to 15 µg mL-1 for all analytes in both biological samples. Intermediate precision was found to be <8.4% over the tested concentration ranges. A run time of <5 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application for the analysis of metronidazole and fluconazole in both breast milk and human plasma and it can be used to support a wide range of clinical studies.