Salting-out Liquid-liquid Extraction Research Articles

Applicability of Salting-Out Liquid-Liquid Extraction for New Fluorimetric Quantification of Omadacycline in Pharmaceutical Formulations and Biological Samples

Objective: A detectable innovative fluorimetric method was used to determine OMC in human plasma matrices, pharmaceutical tablets, and vials with high recovery rates and without biological interference. Method: The fluorimetric technique was used based on the interaction between 4-chloro7-nitrobenzofurazan (NBD-Cl) with a (2-ry amine group) in OMC with pH 8.0, which generates a fluorescent compound measured at 530 nm (exci 470 nm) following a 10-minute heating step at 80 oC. The plasma and milk samples were treated with ammonium sulfate as a salting-out procedure. Results: Omadacycline (OMC) was successfully determined in pharmaceutical, plasma, and milk samples with a linear range from 60.0 to 700.0 ng mL-1, with the lower limit of detection (LOD 5.18 ng mL-1) and limit of quantitation (LOQ 15.72 ng mL-1). Conclusion: This simple, reliable, and detectable fluorimetric method was successfully developed to determine omadacycline in pharmaceutical tablets, plasma samples, and milk with high recovery rates.

Assessing human exposure to pesticides and mycotoxins: optimization and validation of a method for multianalyte determination in urine samples

Humans are exposed to an increasing number of contaminants, with diet being one of the most important exposure routes. In this framework, human biomonitoring is considered the gold standard for evaluating human exposure to chemicals. Pesticides and mycotoxins are chemicals of special concern due to their health implications. They constitute the predominant border rejection notifications for food and feed in Europe and the USA. However, current biomonitoring studies are focused on a limited number of compounds and do not evaluate mycotoxins and pesticides together. In this study, an analytical method has been developed for the determination of 30 pesticides and 23 mycotoxins of concern in urine samples. A salting-out liquid–liquid extraction (SALLE) procedure was optimized achieving recoveries between 70 and 120% for almost all the compounds and limits as lower as when QuEChERS was applied. The compounds were then determined by liquid chromatography coupled to triple quadrupole mass spectrometry. Different chromatographic conditions and analytical columns were tested, selecting a Hypersild gold aQ column as the best option. Finally, the method was applied to the analysis of 45 urine samples, in which organophosphate and pyrethroid pesticides (detection rates (DR) of 82% and 42%, respectively) and ochratoxin A and deoxynivalenol (DR of 51% and 33%, respectively) were the most detected compounds. The proposed analytical method involves the simultaneous determination of a diverse set of pesticides and mycotoxins, including their most relevant metabolites, in human urine. It serves as an essential tool for biomonitoring the presence of highly prevalent contaminants in modern society.Graphical

Groundwater monitoring and leaching of sulfonylurea herbicides and transformation products

Screening of sulfonylurea herbicides (SUHs) in water is important due to their widespread usage and residual activity. Thus, it is essential to develop simple, sensitive and cost-effective pre-treatment method with minimal matrix interferences for quantification of SUHs from water. In present study, sample pre-treatment parameters using salting out liquid liquid extraction (SALLE) method for determining SUHs and their transformation products (TPs) were optimised for improved accuracy and selectivity. A mathematical model representing relationship between the variables and response (percent recovery) was developed using Box behnken design. The developed method has mean percent recovery of 89.80 to 101.20% with relative standard deviation < 10%. Based on greenness evaluation tools viz. Analytical Eco-Scale, GAPI and AGREE metrics, developed SALLE method falls under the category of green analytical methods. To determine the probability of contamination of groundwater by studied herbicides, leaching studies were carried out. About 0.035 ± 0.011 to 0.11 ± 0.013, 0.012 ± 0.014 to 0.042 ± 0.015, 0.009 ± 0.012 to 0.031 ± 0.011, 0.009 ± 0.012 to 0.015 ± 0.011 and 0.01 ± 0.014 to 0.017 ± 0.013 µg mL−1 residues of iodosulfuron methyl, metsulfuron methyl, ethoxysulfuron, 2-amino-4,6-dimethoxypyrimidine and 2-amino-4-methoxy-6-methyl-1,3,5-triazine, respectively were detected in leachates indicating their high potential to contaminate ground water resources. The herbicide residues in real water samples were below permissible limit (<0.01 µg g−1) imposed by World Health Organization and European Protection Agency for drinking water quality.

Bioaccumulation of mycotoxins in human forensic liver and animal liver samples using a green sample treatment

The investigation of the mycotoxin bioaccumulation in human and animals is of wide relevance due to the potential toxicity associated with these secondary metabolites. This study proposes an analytical methodology consisting of salting-out liquid–liquid extraction (SALLE) followed by dispersive liquid–liquid microextraction (DLLME) for the determination of 13 mycotoxins: aflatoxins (G1, G2, B1 and B2), enniatins (A, A1, B and B1), beauvericin, HT-2 and T-2 toxins, zearalenone and deoxynivalenol, in human and animal liver. A targeted analysis was performed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS), as well as the screening of derived metabolites by ultrahigh performance LC and high-resolution mass spectrometry (UHPLC-HRMS). The proposed method was in-home validated, and trueness was verified by apparent recovery studies with values between 94 and 110 %. Furthermore, suitable linearities were obtained by the proposed method for all the mycotoxins and detection and quantification limits allow the mycotoxin monitoring at the low levels expected in biological samples. Repeatability and intermediate precision were calculated at two concentration levels, obtaining values of relative standard deviation below 9.5 %. The proposed methodology allowed to study the bioaccumulation of mycotoxins in both human and animal liver, demonstrating the presence of emergent mycotoxins in all liver samples analyzed, specifically enniatins B, B1 and beauvericin were detected with concentrations up to 4.04 µg kg−1.

Automated centrifugation-less milk deproteinization and homogenous liquid-liquid extraction of sulfonamides for online liquid chromatography

A novel approach to the determination of sulfonamides in milk based on the Lab-In-Syringe technique is presented. The method involves automated salting-out liquid-liquid extraction of the analytes, allowing simultaneous sample deproteination without requiring centrifugation or manual sample handling. The procedural parameters, including salt type, solvent-to-sample ratio, and salt solution volume, were studied. The extracts obtained were diluted in situ and transferred to online coupled liquid chromatography for analyte separation carried out in parallel to the subsequent extraction. In this way, a sample throughput of approximately 6 h−1 was achieved. The detection limits ranged from 25 to 32 μg L−1 using a 500 μL milk sample and spectrophotometric detection. The applicability of the developed method to sample analysis was proven by recovery values ranging from 73.2% to 94.1% (86.3% on average) for milk samples of different fat content spiked at 3 μg mL−1 level. Straightforward automation of one of the most laborious preparation steps in food analysis, i.e., deproteination, was demonstrated.

Novel method for determination of methoxphenidine and its main metabolite in biological samples

Methoxphenidine (MXP) is new psychoactive substance belonging to the group of dissociative anesthetics. It appeared on the black market in 2013, and MXP intoxications have been reported with at least three deaths since then. To understanding the mechanism of toxicity and effects of MXP, it is important to know its pharmacological properties (e.g. toxicity, activity on receptors and ADME) and metabolism which may be further exploited to study its mechanism of action in vivo. Metabolism of such compound may be useful not only in toxicological screenings, but together with pharmacokinetic and pharmacodynamic data it enhances the knowledge how these substances may affect the living organism. It is still very little information on the MXP and a method for analysis of biological samples (e.g. brain, liver, lungs and serum) remain to be developed. Standards of MXP, main metabolite norMXP and internal standard of deuterated MXP (MXP-d3) was prepared in-house and used as external calibrators. For determination of MXP and norMXP in biological samples, UHPLC-MS was used. Quantitative analyses were performed using LC system Dionex Ultimate 3000 (Thermo-Fisher, MA, USA) and the chromatographic separation was performed using Poroshell 120, Phenyl Hexyl (2.1 × 100 mm, 2.7 μm) column (Agilent, CA, USA). Mobile phases consisted of 0.1% formic acid and 10mM ammonium formate in 5% methanol in water (v/v) (A) and in 5% acetonitrile in methanol (v/v) (B) and were used at a flow rate of 0.4mL/min. The analytes were detected in multiple reaction monitoring (MRM) mode using QTRAP 6500+ (AB Sciex, MA, USA) with a Turbo V Ion source operating in positive electrospray (ESI + ) mode. Wistar rats given 20 mg/kg MXP were used as real samples. At 0; 0.5; 1; 2; 4; 8 and 24 hours after administration, samples of livers, lungs, brain and 2 mL of blood serum were collected from rats. There were 8 samples from each tissue at each time point. To extract analytes from these complex biological matrices, protein precipitation and salting-out liquid-liquid extraction (SALLE) were used. Due to the complex matrix, it was important to develop an extraction method with sufficient yield and low matrix effect. Liquid-liquid extraction, SALLE and protein precipitation were tested for each type of biological sample separately. These methods were selected on the basis of a previous study of structurally similar substances. Protein precipitation was chosen as the best extraction method for processing blood serum samples and SALLE was chosen for preparation of three other tissues. All validation parameters met the guidelines from the US Food and Drug Administration (FDA). 8 samples of 4 biological samples were analyzed in 7-time intervals. Based on these data, MXP and norMXP concentration curves were generated. A quantification and extraction method for the determination of MXP and the major metabolite norMXP in blood serum, brain, liver and lung samples from Wistar rats was developed and optimized. Pharmacokinetic profiles for MXP and norMXP in all 4 types of biological material used, were established. This work was a part of the research project supported by the Ministry of Health of the Czech Republic (grant No. NU21-04-00307) and also it was supported from the grants of Specific university research–grant No. A2_FCHI_2022_003 and A2_FPBT_2022_023.

Simultaneous determination of 13 nitrosamine impurities in biological medicines using salting-out liquid-liquid extraction coupled with liquid chromatography tandem mass spectrometry

Nitrosamine impurities are being detected in various pharmaceutical products recently. However, no analytical method is provided for biopharmaceuticals. In present work, a salting-out liquid-liquid extraction (SALLE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for quantification of thirteen nitrosamine contaminations in antibody drugs. The method showed excellent linearity over the range of 0.5–5.0 μg/L with LOQ (limit of quantitation) of 0.5 μg/L for targeted nitrosamines. The method was demonstrated to be accurate (recovery in a range of 75.4–114.7 %) and precise (RSD ≤ 13.2 %) for all nitrosamines using spiked samples. Especially, we found that the satisfactory recoveries for N-nitrosomethyl-4-aminobutyric acid (NMBA, 78.0–96.0 %) and 1-methyl-4-nitrosopiperazine (MeNP, 90.0–109.0 %) were just obtained in the opposite condition (with and without formic acid, respectively). In conclusion, we provide a sensitive and reliable method for nitrosamine estimations to ensure the safety of biological medications.

Sample preparation and antibiotic quantification in vinasse generated from sugarcane ethanol fuel production

Vinasse - liquid organic residue derived from the production of sugarcane ethanol fuel, has been applied as soil amendment via fertigation for decades in Brazil. This organic residue is an important source of nutrients and water for sugarcane crops. Through fertigation, approximately 400 billion liters of vinasse are recycled annually. Despite the economic and agronomic importance of this practice, vinasse-based fertigation can be a source of antibiotic contamination in the environment. The present work reports the application of solid phase extraction (SPE), salting-out liquid-liquid extraction (SALLE), and on-line solid phase extraction (on-line SPE) as sample preparation techniques for the analysis of the following antibiotics (contaminants) in vinasse sample: monensin, penicillin G, virginiamycin M1, virginiamycin S1, tetracycline and erythromycin. The study also employed a totally automated quantitative method based on on-line SPE and liquid chromatography coupled to quadrupole linear ion trap tandem mass spectrometry (LC-QqLIT-MS/MS) for the analysis of these contaminants in vinasse. The application of the aforementioned sample preparation techniques led to the successful extraction of the analytes, and a comparative analysis of the techniques showed that the on-line SPE technique was the most advantageous among the techniques investigated. The quantitative analytical method applied yielded well-defined chromatographic peaks, working range of 1.0–370.0 ng·mL−1, apparent recovery ranging from 80 to 110% for most compounds, repeatability between 3 and 16%, and limits of detection ranging from1.0 to 10 ng·mL−1. The analysis of six vinasse samples from different ethanol producing plants led to the detection of monensin at the concentration of 14.3 ng·mL−1 in their compositions. The results obtained show that fertigation with vinasse is a source of antibiotic contamination in the environment.

Rethinking Drug Analysis in Health Care: High-Throughput Analysis of 71 Drugs of Abuse in Oral Fluid Using Ion Mobility--High-Resolution Mass Spectrometry.

We have identified a clinical need for a sensitive, specific, flexible, comprehensive and affordable analytical technology to efficiently detect polydrug use. In addition, the current standard practice of surveilled urine sampling is uncomfortable for the patient; hence, more patient-friendly sample collection methods are requested. To fill these needs, we have developed and validated a high-throughput liquid chromatography-high-resolution mass spectrometry (LC--HRMS) method for the analysis of drugs of abuse (DoA) in oral fluid (OF). The method covers a panel of 71 substances including traditional DoA, prescription narcotics and new psychoactive substances (NPS), with a guaranteed limit of identification of <3 µg/L for 87% of the analytes. Method validation showed high accuracy (>99.7%), sensitivity (>99.7%) and specificity (100%). Most analytes had a high process efficiency during the salting-out liquid-liquid extraction sample preparation and no or only a minor matrix effect during the analysis. We have implemented this method in clinical routine and present data from 18,579 OF samples collected during routine patient treatment in mainly psychiatric and addiction clinics in West Sweden between September 2020 and June 2021. Seventy-one percent of the samples were positive and a total of 41,472 DoA findings were detected. Amphetamine (27%), buprenorphine (25%), nordiazepam (18%) and alprazolam (16%) were most prevalent. New psychoactive substances were detected in 189 samples (1.0%). The occurrence of polydrug use was common; 34% of the positive samples contained three analytes or more and 12% six or more. To the best of our knowledge, this is the first method for comprehensive analysis of DoA in OF using LC--HRMS and the largest dataset published on the detection of DoA in OF. With the current complex and variable drug use pattern, this broad, cost-effective and reliable method has largely replaced immunoassay screening in urine in our laboratory.

Microwave-assisted solid-phase synthesis of nitrogen-doping carbon dot with good solvent compatibility and its sensing of sunitinib.

Microwave-assisted solid-phase synthesis method was simple, convenient, and fast, and herein adopted to produce nitrogen-doping carbon dots (N-CDs) in only 3min. The N-CDs possessed high fluorescence quantum yield up to 15.9% with satisfactory stability to the environmental pH, ionic strength, and ultraviolet radiation. Particularly, the N-CDs had excellent dispersibility in both water and water-compatible organic solvents with similar fluorescence properties. Sunitinib, a small-molecule tyrosine inhibitor effective for some solid tumors, was found to quench the fluorescence of N-CDs in these media via the inner-filter effect. Hence, it was convenient to combine the proper sample pretreatment with the N-CD probe for sensing sunitinib avoiding the medium incompatibility problem. For rat plasma sample, salting-out liquid-liquid extraction was employed to minimize the sample matrix and concentrate the target sunitinib from aqueous to acetonitrile. The fluorescence detection of sunitinib was then achieved in acetonitrile by the addition of the proper amount of N-CDs. The method provided a good linearity of 0.1μg/mL to 7μg/mL with a limit of detection of 30ng/mL, which met the requirement of the therapeutic drug monitoring of sunitinib. The developed method was potential for on-site detection of sunitinib.

A Salting-out Liquid-Liquid extraction (SALLE) for the analysis of caprolactam and 2,4-di-tert butyl phenol in water and food simulants. Study of the salinity effect to specific migration from food contact materials

Caprolactam and 2,4-di-tert-butyl phenol (2,4-DTBP) are substances typically found in some food contact materials (FCMs). They are known to often migrate into food, and are difficult to analyse in liquid food simulants using GC. In this work a simple salting-out Liquid-Liquid Extraction (SALLE) for the analysis of both substances in water and the official food simulant A (10 % v/v ethanol, Regulation (EU) No. 10/2011) is presented. The method, which included analytical determination by GC-MS, was optimized and validated to ensure sufficient analytical quality.The method’s LOQs allowed the proper quantification of caprolactam at its EU legislative limit (15 mg kg−1). For 2,4-DTBP the method also revealed good sensitivity, although no official limits have been established yet. Linear regression coefficients (R2) were in all cases higher than 0.999, and recoveries ranged from 87 % and 95% for caprolactam and 2,4-DTBP, respectively. Precision was also acceptable, with the RSDs (%) below 12 %. The method proved to be adequate to be used for routine analysis.The presence of salt during migration of caprolactam and 2,4-DTBP was also investigated in this work. Polyamide/polyethylene FCM multilayer films have been tested with water and simulant A, containing different amounts of NaCl (up to 15 % m/v), and applying different migration conditions (temperature and time). The results indicated that salinity plays an important effect on the migration of caprolactam, with the presence of salt reducing its migration in case of water and increasing it in case of simulant A. These preliminary results seem to indicate that migration testing should consider not only the well-known fatty content of a food, but also its salinity content, as it may end up affecting drastically the migration of polar substances.

Occurrence of Organochlorine Pesticides in Human Tissues Assessed Using a Microextraction Procedure and Gas Chromatography-Mass Spectrometry.

This work focuses on the development, validation and application of an analytical method for the determination of twenty organochlorine pesticides (OCPs) in human tissues using salting-out liquid-liquid extraction and dispersive liquid-liquid microextraction for sample preparation and gas chromatography-mass spectrometry to analyze the obtained extracts. Measurement of the concentration levels of these toxics in tissues can be used to assess the risk of the population to exposure. The linearity of the proposed method was verified in the 10-1,000ng/g range. The sensitivity was evaluated calculating the limits of detection (LODs) for 20 OCPs (α-, β-, γ- and δ-hexachlorocyclohexane (HCH), α- and β-endosulfan, endosulfan sulfate, aldrin, dieldrin, endrin, endrin ketone, endrin aldehyde, α- and γ-chlordane, 4,4'-dichlorodiphenyltrichloroethane, 4,4'-dichlorodiphenyldichloroethylene (DDE), 4,4'-dichlorodiphenyldichloroethane, heptachlor, heptachlor epoxide and methoxychlor), most of them being found between 1.0 and 16ng/g. The intra- and interday precisions were <12% for relative standard deviation values. The accuracy of the method was evaluated by recovery studies, which gave recovery percentages in the 85-109% range. Seven different tissues (liver, kidney, heart, spleen, lung, brain and abdominal fat) from eight autopsies were analyzed, and only three cases were seen to have β-HCH and 4,4'-DDE in abdominal fat, while 4,4'-DDE was also detected in the heart of one case. The rest of the samples were free of the studied OCPs at least above the corresponding LODs.

Migration of dihydroxyalkylamines from polypropylene coffee capsules to Tenax® and coffee by salt-assisted liquid–liquid extraction and liquid chromatography–mass spectrometry

Migration of N,N-Bis(2-hydroxyethyl) alkyl(C8–C18)amines from five different polypropylene capsules to Tenax® and coffee powder have been studied. A single step extraction-cleanup procedure using salting out liquid–liquid extraction (SALLE) method followed by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was applied. The critical parameters on the SALLE procedure such as extracting solvent, extracting volume, sample pH, salt and its concentration were optimized. The recovery values were in the range of 87.5%–106.5%. The %RSD were lower than 3.7%. The limit of detection was improved from 2.3 ng/g in Tenax® to 0.8 ng/g in coffee. The results indicated that the analyzed compounds have the potential to migrate from the polypropylene capsule containers to the coffee. In most of the cases, the migrated values were higher in Tenax® than in coffee in a range between 1.8 and 61%. One sample did not comply with the specific migration limit established by the European Commission.

Determination of carbazole and halogenated carbazoles in human serum samples using GC-MS/MS

Carbazole and halogenated carbazoles have been widely detected throughout the environment in soil, river deposits, and lake sediments. Human exposure to these compounds may occur through inhalation, drinking water, dietary intake and/or skin contact, and exposure levels in the body may be evaluated by measuring them in serum or blood. This paper reports the method development and validation for the analysis of carbazole and 11 halogenated carbazoles in human blood and/or serum samples. A small sample size of 100 μL of blood or serum was employed for the analysis. The samples were prepared through salting-out liquid-liquid extraction (LLE) by using hexane/ethyl acetate (4:1, v/v) as the extraction solvent and aqueous MgSO4 (37.5 wt%) as the salting-out regent, respectively. Sample analysis was performed using gas-chromatography (GC) coupled with a tandem mass spectrometer (MS/MS) in an electron impact (EI) mode. The developed method demonstrated low detection limits in the range of 0.02–0.27 ng/mL, intra-day accuracy ranging from 81.2% to 125%, and inter-day accuracy from 91.0% to 117%. The intra- and inter-day precisions, calculated by relative standard deviations (RSDs), were in the ranges of 1.0–16.0% and 1.8–16.4%, respectively. The developed method was applied to the analysis of 50 human serum samples collected from pregnant women in Southern California in 2012. Low concentrations of carbazole were measured in 18 samples, while halogenated carbazoles were not detected in any of the samples.

Trace-level determination of two neonicotinoid insecticide residues in honey bee royal jelly using ultra-sound assisted salting-out liquid liquid extraction followed by ultra-high-performance liquid chromatography-tandem mass spectrometry

A few studies have investigated the analysis of neonicotinoid insecticides in honey bee royal jelly. An analytical method based on salting-out liquid-liquid extraction assisted with ultrasound followed by liquid chromatography-tandem mass spectrometry was developed for the determination of thiamethoxam and clothianidin in royal jelly. A cleaning step based on freezing before injection to the UHPLC-MS/MS (MRM mode) allowed maximizing the signal-to-noise ratio. The finalized method was validated in line with European legislation. The limits of quantification (LOQ), reached from a test sample of 100 mg royal jelly only, was 0.25 ng/g for both neonicotinoids. The recoveries were close to 100% with relative standard deviation below 13.7%, measured at three concentration levels. The validated method was finally applied to samples of royal jelly commercially available, and showed the presence of thiamethoxam at levels between the limit of determination (LOD = 0.15 ng/g) and LOQ in a few samples.

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) - a group of new psychoactive substances (NPS) - is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and finally included to the MS/MS spectra in-house library. Sample preparation was performed using a cheap-and-easy salting-out liquid-liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.

Monitoring of cyanotoxins in water from hypersaline microalgae colonies by ultra high performance liquid chromatography with diode array and tandem mass spectrometry detection following salting-out liquid-liquid extraction

In this study two different analytical approaches have been developed to determine the presence of several cyanotoxins in saline water samples from a continental salt marsh. A salting-out assisted liquid-liquid extraction (SALLE) has been used in combination with ultra-high performance liquid chromatography-tandem mass spectrometry and UV-diode array detection (UHPLC-MS/MS and UHPLC-DAD). The target analytes are eight microcystins named MC-RR, MC-YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, MC-LF and nodularin (NOD), covering a wide range of polarities. The separation was achieved using a Zorbax Eclipse Plus RRHD C18 column (50 × 2.1 mm, 1.8 μm) in less than 7.5 and 5.5 min for UV and MS/MS detection, respectively. The mobile phase used consisted of water (solvent A) and acetonitrile (MeCN) (solvent B), both containing 0.01% of formic acid for DAD and 0.4% of formic acid for MS/MS detection, at a flow rate of 0.4 mL min−1. The temperature of the column was set at 25 °C and 20 μL of sample were injected. The main parameters affecting the SALLE procedure were studied and the following optimum values were obtained: neutral pH, 2 mL of acetonitrile as extraction solvent and 1.2 g of ammonium sulfate as salting-out agent for 4 mL of water sample. The validation protocols for both methods were accomplished with real water samples obtaining LODs ranging from 1.0 to 3.4 μg L−1 and 0.02 to 0.11 μg L−1 for DAD and MS/MS respectively. Although the SALLE-UHPLC-DAD methodology is easier and cheaper than UHPLC-MS/MS significantly better detection limits were achieved with tandem mass spectrometry as well as allowing for unambiguous identification. Extraction recoveries were higher than 77.0% (except for MC-RR and NOD which were 53.2% and 54.3, respectively) with satisfactory inter-day and intra-day precisions (RSD below 13.3%).

Spectrophotometric determination of phenol and chlorophenols by salting out assisted liquid-liquid extraction combined with dispersive liquid-liquid microextraction.

In this work, salting out liquid-liquid extraction (SALLE) combined with dispersive liquid-liquid microextraction (DLLME) was developed as a novel extraction method for extraction and preconcentration of phenol and chlorophenols (CPs) in environmental water samples. The analytes were derivatized with 4-aminoantipyrineand determined by spectrophotometry. Experimental parameters such as type and volume of the organic solvent, type and amount of salt, pH and vortex time were optimized. Under the optimum conditions, calibration curves were linear in the range of 1-300 μg L-1 and limit of detections (LODs) were in the range of 0.15-0.22 μg L-1. The extraction recoveries and enrichment factors ranged from 94.80% to 106.1% and 78.12 to 82.53, respectively. Repeatability of method based on five replicate measurements of phenols was in the range of 4.8-7.2%. The results obtained in this study showed that the proposed method is simple, rapid and environmentally friendly with high extraction efficiency for preconcentration and determination of phenol and CPs in real samples. The proposed method was also compared with the reference method.

Urinary levels of enniatin B and its phase I metabolites: First human pilot biomonitoring study

Enniatins (Enns) are mycotoxins produced by Fusarium spp. and are widely distributed contaminants of cereals and derivate products. Among the different identified enniatins, Enn B is the most relevant analogue in cereals in Europe. Therefore, the aim of this study was to investigate for the first time the occurrence of Enn B and Enn B phase I metabolites in 300 human urine samples throughout an ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) methodology. Three different sample preparation procedures were evaluated and salting-out liquid-liquid extraction showed satisfactory validation results. Enn B was quantified in 83.7% of samples ranging from 0.006 to 0.391 ng/mL (average content: 0.016 ng/mL). In line with the in vitro observations with human liver microsomes, in the here analyzed samples the Enn B monooxygenated, N-demethylated and dioxygenated metabolites were tentatively found in 87.7%, 96.3% and 6.7% of samples. The data of this pilot biomonitoring survey indicate a frequent intake of enniatins in Italy, supporting further toxicological studies to provide better basis for understanding their potential effects in humans.

In-house validation of a rapid and efficient procedure for simultaneous determination of ergot alkaloids and other mycotoxins in wheat and maize.

A fundamental step in addressing the global problem of mycotoxins is the development of highly sensitive, multi-class extraction and detection methods. This constitutes a field of research that has in recent years enjoyed a steady advance. Such methods, generally based on liquid chromatography coupled to mass spectrometry, are widely reported successfully detecting various mycotoxins in different food and feed samples. In this work, an innovative approach to multi-class mycotoxin control is proposed, offering specific advantages: a broader inclusion of more mycotoxin classes, robust and thorough extraction for all target compounds despite their varied chemical properties, and determination of all analytes from a single injection. The method involved the extraction and quantification of the main mycotoxins produced by Aspergillus, Fusarium, and Penicillium fungi, as well as their reported derivatives, together with 12 other compounds most commonly produced by Claviceps purpurea. The popularly reported QuEChERS technique has been reduced to a simple "salting-out liquid-liquid extraction" (SO-LLE) to obtain the most efficient extraction of the aforementioned mycotoxin classes in a very short time. This is in particular extremely important in ensuring correct determination of individual ergot alkaloids, for which short and robust sample preparation as well as short analytical sequences were key for minimizing the epimerization during analysis. The analyses of wheat and maize samples were performed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Matrix-matched calibration curves were established and limits of quantification were below the maximum levels established by the EU regulation. The precision (repeatability and intermediate precision) was lower than 13% in all cases and recoveries ranged between 60 and 98% in maize and between 62 and 103% in wheat, fulfilling the current legislation. The method was applied to study the co-occurrence of these mycotoxins in wheat (n= 13) and maize (n= 15) samples from six European countries. A successful quantification of 23 different mycotoxins, from all major classes, in 85% of wheat and 93% of maize samples was achieved.