Ultrasound assisted membrane-assisted solvent extraction for the simultaneous assessment of some drugs involved in drug-facilitated sexual assaults by liquid chromatography-tandem mass spectrometry.

A simple and highly efficient ultrasound assisted membrane-assisted solvent extraction (MASE) pre-treatment method for urine has been developed and validated for the simultaneous determination of twenty-two drugs involved in drug-facilitated sexual assaults (DFSAs) by liquid chromatography-tandem mass spectrometry. MASE was performed with 4.0 mL of urine (pH adjusted at 12), 400 µL of hexane as an organic solvent inside the polypropylene membrane, and ultrasonication (45 kHz, 120 W) for 10 min. A pre-concentration factor of 40 was achieved after evaporation (N2 stream) and re-dissolution in 100 µL of methanol. Analytes were separated using a Zorbax Eclipse Plus C18 column under gradient elution with aqueous 10 mM NH4HCO3 (pH 8.0) and methanol as mobile phases. Matrix-matched calibrations allowed the assessment of DFSA drugs of quite different octanol-water partition coefficients (Ko/w), from 1.32 101 for pregabalin to 2.45 105 for clomipramine (Log P values from 1.12 (pregabalin) to 5.39 (clomipramine)). The limit of detection (LOD) was between 0.0075 to 0.37 µg L-1, with analytical recoveries ranging from 73 to 103%, and relative standard deviations (RSDs) within the 2-20% range. The applicability of the method was demonstrated after analysing urine samples under forensic investigation.

Bioavailability of Aflatoxins in Cultured Fish and Animal Livers Using an In Vitro Dialyzability Approach.

The objective of the present study was to investigate the bioavailability of aflatoxins (AFs) from fish, and chicken and rabbit livers using an in vitro dialyzability approach. Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to assess the aflatoxin content in samples, as well as in dialyzate and residue fractions after the in vitro procedure. A vortex-assisted dispersive liquid-liquid microextraction (VALLME) technique was used for preconcentrating AFs before determination. Raw samples showed bioavailability ratios of 41-45% for aflatoxin B1 (AFB1), 28-38% for aflatoxin B2 (AFB2), and 42% for aflatoxin G2 (AFG2). Aflatoxin G1 (AFG1) was not detected. The culinary process (steaming or grilling) was found to change AFs' bioavailability (higher bioavailability ratios were found in cooked samples). AFB2 was found to be transformed into other compounds during the in vitro process, and the presence of AFB2 and AFB2 transformation/degradation products was investigated and confirmed by high-resolution mass spectrometry (HRMS).

Molecularly Imprinted Polymer for a Smart Dispersive Micro-Solid Phase Extraction Technique for Assessing Trace Level Aflatoxins in Cultured Fish.

Molecularly imprinted technology (MIT) consists of preparing materials exhibiting specific recognition cavities to selective mimic the target analytes. The prepared materials promote selective interactions with the targets and avoid interactions of concomitants from complex food, biological, clinical, and environmental matrices. This chapter provides information on a recent development of a vortex-assisted micro-solid phase extraction using a molecularly imprinted polymer (MIP) as an adsorbent for aflatoxins (AFs) determination in cultured fish. MIP particles were synthesized by precipitation polymerization using 5,7-dimethoxycoumarin as a dummy template, methacrylic acid as a functional monomer, divinylbenzene as a cross-linker, and 2,2-azobisisobutyronitrile as an initiator. Polymerization following the precipitation method guarantees homogeneous particle size distribution and the integrity of the imprinted cavities. The MIP microparticles were found to have 5 µm in diameter and a spherical shape. Important parameters such as sample extract pH, adsorption stirring speed and time, desorption stirring speed and time, elution solvent composition and volume, and polymer mass, were fully optimized. The pre-concentration method allows therefore the assessment of four major AFs (B1, B2, G1, and G2) present in cultured fish at very low levels, with pre-concentration factors from 15 to 50 depending of the volume of extract used for performing the dispersive micro-solid phase extraction (D-µ-SPE).

Assessment of trace levels of aflatoxins AFB1 and AFB2 in non-dairy beverages by molecularly imprinted polymer based micro solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A selective molecularly imprinted polymer (MIP) adsorbent was synthesised and used in a batch micro-solid phase extraction format for isolating aflatoxins (AFB1, and AFB2) from non-dairy beverages before liquid chromatography-tandem mass spectrometry determination. MIP synthesis (precipitation polymerization in 3 : 1 acetonitrile/toluene as a porogen) was performed with 5,7-dimethoxycoumarin (DMC), methacrylic acid (MAA) and divinylbenzene-80 (DVB) as a dummy template, functional monomer and cross-linker, respectively (1 : 4 : 20 molar ratio). 2,2'-Azobisisobutyronitrile (AIBN) was used as a polymerization initiator. The adsorbent MIP (50 mg) was enclosed in a cone-shaped polypropylene membrane (porous membrane protected molecularly imprinted micro-solid phase extraction), and parameters such as sample pH, mechanical (orbital-horizontal) shaking, the extraction time (loading stage), the composition of the eluting solution, and the desorption time were optimised. The highest extraction yields were obtained by using 5 mL of non-dairy beverages (pH adjusted at 6.0), and mechanical shaking (150 rpm) for 15 min. Elution was performed with 5 mL of an acetonitrile/formic acid (97.5 : 2.5) mixture under ultrasound (325 W, 35 kHz) for 15 min. After eluate evaporation to dryness and re-dissolution in 150 µL of the mobile phase, the pre-concentration factor of the method was 33.3, which yields limits of detection within the 0.085-0.207 µg L-1 range. In addition, the current proposal was shown to be an accurate and precise method through relative standard deviation of intraday and inter-day assays below 18% and analytical recoveries in the range of 91-104%. However, the method was found to suffer from matrix effects.

Miniaturized vortex assisted-dispersive molecularly imprinted polymer micro-solid phase extraction and HPLC-MS/MS for assessing trace aflatoxins in cultured fish.

A dispersive micro-solid phase extraction approach using a molecularly imprinted polymer as an adsorbent has been developed for pre-concentrating aflatoxins from cultured fish. Aflatoxins were first isolated from fish muscle and liver by an ultrasound assisted extraction procedure using a 60 : 40 acetonitrile/0.1 M KH2PO4 aqueous buffer (pH 6.0) mixture. Polymeric adsorbent beads were synthesized using 5,7-dimethoxycoumarin as a dummy template, methacrylic acid as a functional monomer, divinylbenzene as a crosslinker, and 2,2'-azobisisobutyronitrile as an initiator. Parameters affecting the steps of extraction procedure, including the sample (fish extract) pH, adsorption stirring speed and time, desorption stirring speed and time, elution solvent ratio, and polymer capacity, were investigated and optimized. The limit of detection was found to vary from 0.29 to 0.61 µg kg-1 for the several aflatoxins. The proposed method was shown to be accurate and precise. Intraday and inter-day relative standard deviations were lower than 20%, and intraday and inter-day analytical recoveries were within the 80-100% range. The prepared adsorbent in the dispersive micro-solid phase extraction format was re-usable, and the pre-concentration procedure was found to be simple, rapid and highly selective and sensitive to identify/quantify AFs in fish.

Combining ultrasound-assisted extraction and vortex-assisted liquid-liquid microextraction for the sensitive assessment of aflatoxins in aquaculture fish species.

Although aflatoxins contamination in feedstuff is a well-known problem, and hence these residues are controlled in poultry products, there is scarce information regarding the presence of these toxic substances in aquaculture fish, facilities that use several feedstuff for fish breeding. A simple, rapid, and sensitive method has been therefore developed for aflatoxins (B1, B2, G1, and G2) assessment in aquaculture products by combining ultrasound probe-assisted extraction and vortex-assisted liquid-liquid microextraction as a sample pretreatment, and high-performance liquid chromatography-tandem mass spectrometry as a separation/detection system. Aflatoxins were extracted from fish flesh/liver with a 60:40 acetonitrile/aqueous phosphate buffer (pH 7.0) mixture before preconcentration and clean-up by vortex-assisted liquid-liquid microextraction under the following optimized conditions: 5.0 mL of fish extract at pH 7.0 and NaCl at 0.5% (w/v), 400 µL of chloroform as extracting solvent, and vortex shaking at 2000 rpm for 1 min. The proposed method is shown to be precise and accurate, and the limit of quantitations (from 0.20 to 1.10 µg kg-1 ) were lower than the value established by the European Commission Regulation for aflatoxins in foodstuff. Results have shown that fish flesh is free of aflatoxins, but aflatoxins B2 and G1 were quantified in fish liver.

Ultrasound assisted combined molecularly imprinted polymer for the selective micro-solid phase extraction and determination of aflatoxins in fish feed using liquid chromatography-tandem mass spectrometry.

A combined procedure based on using ultrasounds for target isolation followed by porous membrane-protected micro solid phase extraction using a molecularly imprinted polymer as an adsorbent has been developed as a highly selective extraction and clean-up procedure for isolating aflatoxins B1, B2, G1, and G2 from fish feed before ultra-high-performance liquid chromatography tandem mass spectrometry determination. Polymeric adsorbent beads have been synthesized by the precipitation polymerization method which guarantees a homogeneous particles size distribution and the integrity of the generated imprinted cavities. In addition, polymerization was performed using a higher proportion of organic solvent (toluene) in the porogen mixture, which generates MIP particles adequate for interacting with targets dissolved in organic (hydro-organic) mixtures (extracts from fish feed). These approaches led to a selective and high efficient pre-concentration method for AFs. Ultrasound-assisted extraction (10 mL of 60:40 acetonitrile/0.1 M KH2PO4 pH 6.0, 40% amplitude, continuous sonication for 7.0 min) allowed an efficient aflatoxins isolation from fish feed. In addition, the resulting pH of the extract (pH 7.0) has been found to be the optimum for performing clean-up/pre-concentration (enrichment factor of 33.3) by molecularly imprinted polymer based micro-solid phase extraction (orbital horizontal shaking speed at 150 rpm for 10 min for loading, and 5 mL of 95:5 acetonitrile/formic acid as eluting solution using ultrasounds 35 kHz for 15 min). The current proposal was shown to be an accurate and precise method through relative standard deviation of intraday and inter-day tests below 20% and analytical recoveries in the range of 80-100%. The limits of detection were within the 0.42-1.15 µg kg-1 range, quite lower than those established by European Commission guidelines for aflatoxins in animal feeds.