Simple Sample Preparation Method Research Articles

Simultaneous Quantification of Nine Antimicrobials by LC-MS/MS for Therapeutic Drug Monitoring in Critically Ill Patients.

Adequate antibiotic treatment is a prerequisite for the successful treatment of systemic infections. Based on accumulating scientific evidence, a fixed dosage regimen can lead to insufficient and ineffective antibiotic therapy. Thus, the aim of this study was to develop and validate a simplified, but sensitive method for the simultaneous quantification of antimicrobials by using liquid chromatography with tandem mass spectrometry (LC-MS/MS) for the development of personalized therapy regimens using therapeutic drug monitoring. A method was developed for the simultaneous quantification of 9 antimicrobials (aciclovir, ampicillin, cefuroxime, ciprofloxacin, meropenem, metronidazole, piperacillin, rifampicin, and tazobactam) in lithium-heparin plasma. A simple sample preparation method and a chromatographic run time of 10 minutes enabled the quick processing of the samples. The method was validated according to the guidelines for bioanalytical method validation of the European Medicines Agency and addressed sensitivity, specificity, linearity, accuracy, precision, dilution integrity, carry-over, recovery, matrix effects, and stability. The chromatographic run time was 10 minutes and antimicrobials eluted at retention times ranging from 1.1 to 2.2 minutes. Calibration curve for all antimicrobials was linear over a range of 1-100 mg/L, and a 2-fold or 5-fold dilution of the samples was possible. The method accuracy ranged from 85.1% to 114.9% for all measured antimicrobials, and the within- and between-run precision values were <11.9% and <16.5% for the lower limit of quantification. No interferences and carry-over were observed. The samples were stable for at least 5 hours at room temperature or in the autosampler (10°C). The LC-MS/MS method developed in this study is appropriate and practical for the therapeutic drug monitoring of antimicrobials in the daily clinical laboratory practice because of its short analysis time, the need for a small amount of plasma, high specificity, and accuracy.

An enhanced sensitivity and cleanup strategy for the nontargeted screening and targeted determination of pesticides in tea using modified dispersive solid-phase extraction and cold-induced acetonitrile aqueous two-phase systems coupled with liquid chromatography-high resolution mass spectrometry

At present, matrix interferences in tea are still a great challenge for analysis of multi-pesticide residues. Herein, a simple sample preparation method was developed based on the modified dispersive solid-phase extraction (DSPE) procedure and cold-induced acetonitrile aqueous two-phase systems (ATPS). In modified DSPE procedure, polyvinylpolypyrrolidone (PVPP) was selected as the matrix dispersive adsorbent to remove polyphenols from the tea with the removal rate of >98.3%. Using cold-induced acetonitrile ATPS, caffeine of the tea extract was sharply reduced by 81.2%, and a 0.798–3.167 of enrichment factor for the representative pesticides was achieved. In liquid chromatography-high resolution mass spectrometry analysis, a full scan/data independent acquisition approach was used for the nontargeted screening and targeted determination of pesticides. In data analysis, an in-house database of pesticides was constructed, and a simple accurate mass calibration method was used to correct the accurate mass variation, which was conducive to reduce the number of false detects.

Determination of selected emerging contaminants in freshwater invertebrates using a universal extraction technique and liquid chromatography accurate mass spectrometry.

A simple sample preparation method based on a modified liquid-phase extraction approach to extract selected pharmaceuticals and personal care products from freshwater organisms is described. Extracted samples were analysed using liquid chromatography with Q-Exactive plus hybrid quadrupole Orbitrap mass spectrometry, using 2.6 μm C18 media. A 0.1% v/v acetic acid/acetonitrile mobile phase was applied over a 20min gradient. Method detection limits in full scan mode were ca. 0.04-2.38ng of analyte per g of sample. Linearity ranged from 0.9750 to 0.9996 over the calibration range of 0.01-100μg/L; MS mass accuracy was <2ppm for most analytes. This method was applied to quantify six pharmaceuticals and personal care products in seven invertebrate samples. For tandem mass spectrometry analysis, selection of precursor ions was performed for each pharmaceutical, with Mass Frontier software illustrating the fragmentation mechanism. Effects of collision energy on intensities of ions was further investigated. The tandem mass spectrometry condition resulting in the highest signal of respective selected product ion was selected to confirm each pharmaceutical, which was initially observed in the full scan mode. Results indicate that pharmaceuticals and personal care products found to be present in water-ways, may be incorporated into organisms that live in the environment of affected water streams.

Determination of Penicillins G and V in Vegetables and Fruits by Exact Masses of Ions of Protonated Adducts with Methanol by Ultra-High-Performance Liquid Chromatography‒Time-of-Flight High Resolution Mass Spectrometry

A procedure is developed for the rapid screening and sensitive determination of penicillins G and V in vegetables and fruits using ultra-high-performance liquid chromatography‒quadrupole time-of-flight high resolution mass spectrometry for exact masses of ions of protonated penicillin adducts with methanol. A simple and quick method of sample preparation (simplified QuEChERS method) is proposed for the analysis of fruits and vegetables (oranges, lemons, plums, pears, peaches, cabbage, potatoes, onions, carrots, beets, avocados, cucumbers, tomatoes, eggplants). Identification is carried out by the exact ion masses of analytes, retention time, and the pattern of the isotope distribution of ions (mSigma). Because of the significant matrix effect, analysis should be performed by the method of standard additions, by internal standard method (oxacillin), or using matrix grading. The limits of detection were 0.05‒0.3 ng/g, the analytical ranges were 0.2 (0.9)‒10 ng/g (R2 ≥ 0.99), the duration of analysis was 15‒20 min, RSD ≤ 10%.

Use of hydrolytic enzymes as green and effective extraction agents for ultrasound assisted-enzyme based hydrolytic water phase microextraction of arsenic in food samples

In order to contribute to the development and improvement of green sample preparation techniques, a new special approach combining the principles and advantages of microextraction techniques, hydrolytic enzymes and ultrasonic radiation power is presented. This new approach is called as ultrasound assisted-enzyme based hydrolytic water phase microextraction method (UA-EH-WPME). In this study, We developed and used a solvent-free UA-EH-WPME method as innovative, green and simple sample preparation method for the extraction of arsenic (As) in rice and flour samples prior to Inductively Coupled Plasma–Mass Spectrometer (ICP-MS) determination. The UA-EH-WPME method based on the extraction of total As in 10.0 mg of food samples to 300 µL of pH 7.0 aqueous phase with the help of the 3.0 mg of α-amylase in as little as 5 min. In this method, α-amylase acts as a bond breaker agent to break down certain bonds of bio-molecules in food matrix, which lead to extraction of As from food matrix to aqueous phase. 1568A Rice Flour certified reference material was used to optimize the important analytical parameters, which were type of hydrolytic enzyme, pH, volume of aqueous phase, amount of enzyme, temperature of extraction medium and time of ultrasonic radiation, for the quantitative extraction of As from food matrix to aqueous phase. This innovative solvent-free method leads to emerge new ideas in the sample preparation field, by using the benefits of microextraction techniques, hydrolytic enzymes and ultrasonic radiation power, such as elimination of the toxic solvent usage, necessity of mg level of enzyme and food samples, very short and simple extraction process. The LOD, inter-day RSD and intra-day RSD values for the developed UA-EH-WPME/ICP-MS procedure were found as 27.3 µg kg−1, 4.27% and 6.13%, respectively.

A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin.

One of the deadliest mushrooms is the death cap mushroom, Amanita phalloides. The most toxic constituent is α-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were generated and characterized. Both α- and β-amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. Ultimately, these efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays (limit of detection (LOD), ~1.0 µg/L). The assays were characterized for their selectivity and were found to equally detect α-, β-, and γ-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method. This enzyme-linked immunosorbent assay (ELISA) is a simple and fast test, and readily detects amatoxins extracted from A. phalloides.

A sublimate sorbent for stir-bar sorptive extraction of aqueous endocrine disruptor pesticides for gas chromatography-electron capture detection

A dumbbell-shaped magnetic stir-bar with sublimate sorbent was prepared for the stir bar sorptive extraction (SBSE) of pesticides in an aqueous sample prior to gas chromatography-micro-electron capture detection (GC-μECD). Cyclododecane (CDD) was coated onto a magnetic stir-bar surface as a sublimate sorbent, and steel balls were placed on both ends to form a dumbbell-shaped magnetic stir-bar for SBSE. Four EDC pesticides including chlorpyrifos, ethion, bromopropylate, and λ-cyhalothrin in aqueous samples were selected as model species to examine the proposed SBSE and the following desorption.The parameters studied were those affecting the extraction efficiencies including the coating (solvent for CDD and thickness), extraction (sample pH, stirring rate, time, and salting out effect), dissolution solvent volume, and the loss of CDD sublimated in air. The maximum extraction efficiency was obtained under the following conditions. The stir bar (with CDD thickness of 5.2 μm) was added into a 10 mL sample solution (at pH 7) for a 20-min extraction at 600 rpm. Then, the stir bar was gently removed from the sample solution, disassembled, and immersed into a 0.2 mL insert tube consisting of 3 μL hexane to dissolve; 1 μL was used for GC-ECD analysis. The linear ranges were 0.005–5 μg L−1 with coefficients of determination ranging from 0.9950 - 0.9994. Detection limits (based on S/N = 3) of the four EDCs were 0.4–4.5 ngL-1 with a relative standard deviation (RSD) of 2.4–6.3%, and quantitation limits (based on S/N = 5) were 1–15 ngL−1. The relative recoveries of the spiked samples were in the range of 83.2–98.7% with RSDs of 2.1–8.4% in farm field waters. The proposed sublimation sorbent obtained excellent enrichment factors (101–834) and provided a simple, rapid, sensitive, and eco-friendly sample preparation method.

Transmission Kikuchi diffraction from nano-crystalline Ti and TiN thin-films

Nano-crystalline Ti and TiN thin-films fabricated on <100> P-type Si wafers via magnetron sputtering are characterized by transmission Kikuchi Diffraction (TKD) in an FEI Sirion scanning electron microscope (SEM). A simple and cost-effective sample preparation combined with the TKD experiment at working distance of ∼ 3 mm and a tilt of 35° produced indexable Kikuchi patterns. Pole figures generated by automatic and manual indexing revealed <0001>|| normal direction (ND) fiber texture and <111>||ND fiber texture in monolithic Ti and TiN thin-films, respectively. Glancing angle x-ray diffraction results corroborated the texture in the samples. Spatial resolution of ∼ 60 nm is achieved that is close to the limit of resolution in conventional electron backscatter diffraction (EBSD). It is demonstrated that a simple sample preparation method in TKD technique combined with selected area electron diffraction (SAED) in transmission electron microscope (TEM) may reveal fundamental knowledge about different mechanisms occurring during fabrication and processing of the thin-film systems.

Extended investigation of tube-gel sample preparation: a versatile and simple choice for high throughput quantitative proteomics

Sample preparation for quantitative proteomics is a crucial step to ensure the repeatability and the accuracy of the results. However, there is no universal method compatible with the wide variety of protein extraction buffers currently used. We have recently demonstrated the compatibility of tube-gel with SDS-based buffers and its efficiency for label-free quantitative proteomics by comparing it to stacking gel and liquid digestion. Here, we investigated the compatibility of tube-gel with alternatives to SDS-based buffers allowing notably the extraction of proteins in various pH conditions. We also explored the use of photopolymerization to extend the number of possibilities, as it is compatible with a wide range of pH and is non-oxidative. To achieve this goal, we compared six extraction buffers in combination with two polymerization conditions to further optimize the tube-gel protocol and evaluate its versatility. Identification and quantitative results demonstrated the compatibility of tube-gel with all tested conditions by overall raising quite comparable results. In conclusion, tube-gel is a versatile and simple sample preparation method for large-scale quantitative proteomics applications. Complete datasets are available via ProteomeXchange with identifier PXD008656.

Green and simple analytical method to determine benzimidazoles in milk samples by using salting-out assisted liquid-liquid extraction and capillary liquid chromatography

A green and simple multiresidue method using capillary liquid chromatography (CLC) with UV-diode array detection (DAD) has been developed for the determination of sixteen benzimidazoles (BZs) and its metabolites in milk samples. The separation was achieved in <32 min, using a Zorbax XDB-C18 column (150 mm × 0.5 mm I.D, 5 μm), with a mobile phase consisting of 50 mM ammonium acetate (solvent A) and a mixture of acetonitrile/methanol (1:1 v/v) (solvent B), at a flow rate of 9 μL min−1. The temperature of the column was 20 °C and 6 μL of sample were injected. In spite of the complexity of milk samples, an effective, simple and fast sample preparation method called salting out-assisted liquid-liquid extraction (SALLE) was developed for the analysis of these compounds in cow milk samples. To obtain satisfactory extraction efficiencies for the studied analytes, several parameters affecting the SALLE procedure were optimized including the amount of sample, type and volume of the extraction solvent, and the nature and amount of the salt. Good linearity was obtained (R2 > 0.9985 for all BZs) with limits of detection (LOD) between 1.0 and 2.8 μg kg−1. Relative standard deviations of repeatability and intermediate precision were below 1.6 and 14.2%, respectively. Satisfactory recoveries between 79.1 and 99.6% were also obtained for three types of milk samples (cow, sheep and goat). The advantages of a miniaturized technique such as CLC in terms of better efficiencies and reduced solvent consumption, combined with the simplicity of the SALLE procedure, make this method a useful alternative for the monitoring of these residues at trace level.

A method for estimating relative changes in the synaptic density in Drosophila central nervous system

BackgroundSynapse density is an essential indicator of development and functioning of the central nervous system. It is estimated indirectly through the accumulation of pre and postsynaptic proteins in tissue sections. 3D reconstruction of the electron microscopic images in serial sections is one of the most definitive means of estimating the formation of active synapses in the brain. It is tedious and highly skill-dependent. Confocal imaging of whole mounts or thick sections of the brain provides a natural alternative for rapid gross estimation of the synapse density in large areas. The optical resolution and other deep-tissue imaging aberrations limit the quantitative scope of this technique.ResultsHere we demonstrate a simple sample preparation method that could enhance the clarity of the confocal images of the neuropil regions of the ventral nerve cord of Drosophila larvae, providing a clear view of synapse distributions. We estimated the gross volume occupied by the synaptic junctions using 3D object counter plug-in of Fiji/ImageJ®. It gave us a proportional estimate of the number of synaptic junctions in the neuropil region. The method is corroborated by correlated super-resolution imaging analysis and through genetic perturbation of synaptogenesis in the larval brain.ConclusionsThe method provides a significant improvement in the relative estimate of region-specific synapse density in the central nervous system. Also, it reduced artifacts in the super-resolution images obtained using the stimulated emission depletion microscopy technique.

2-Naphthalenthiol derivatization followed by dispersive liquid–liquid microextraction as an efficient and sensitive method for determination of acrylamide in bread and biscuit samples using high-performance liquid chromatography

In this research, an ultrasonic-assisted extraction followed by 2-naphthalenthiol derivatization and dispersive liquid-liquid microextraction of acrylamide (AA) was developed as simple and sensitive sample preparation method for AA in bread and biscuit samples using high performance liquid chromatography. Influence of derivatization and microextraction parameters were evaluated and optimized. Results showed that the derivatization of AA leads to improve its hydrophobicity and chromatographic behavior. Under optimum conditions of derivatization and microextraction, the method yielded a linear calibration curve ranging from 10 to 1000 μg L−1 with a determination coefficient (R2) of 0.9987. Limit of detection (LOD) and limit of quantification (LOQ) were 3.0 and 9.0 μg L−1, respectively. Intra-day (n = 6) and inter-day (n = 3) precisions based on relative standard deviation percent (RSD%) for extraction and determination of AA at 50 and 500 μg L−1 levels were less than 9.0%. Finally, the performance of proposed method was investigated for determination of AA in some bread and biscuit samples, and satisfactory results were obtained (relative recovery ≥ 90%).

Simple Sample Preparation Method for Direct Microbial Identification and Susceptibility Testing From Positive Blood Cultures.

Rapid identification and determination of the antibiotic susceptibility profiles of the infectious agents in patients with bloodstream infections are critical steps in choosing an effective targeted antibiotic for treatment. However, there has been minimal effort focused on developing combined methods for the simultaneous direct identification and antibiotic susceptibility determination of bacteria in positive blood cultures. In this study, we constructed a lysis-centrifugation-wash procedure to prepare a bacterial pellet from positive blood cultures, which can be used directly for identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and antibiotic susceptibility testing by the Vitek 2 system. The method was evaluated using a total of 129 clinical bacteria-positive blood cultures. The whole sample preparation process could be completed in <15 min. The correct rate of direct MALDI-TOF MS identification was 96.49% for gram-negative bacteria and 97.22% for gram-positive bacteria. Vitek 2 antimicrobial susceptibility testing of gram-negative bacteria showed an agreement rate of antimicrobial categories of 96.89% with a minor error, major error, and very major error rate of 2.63, 0.24, and 0.24%, respectively. Category agreement of antimicrobials against gram-positive bacteria was 92.81%, with a minor error, major error, and very major error rate of 4.51, 1.22, and 1.46%, respectively. These results indicated that our direct antibiotic susceptibility analysis method worked well compared to the conventional culture-dependent laboratory method. Overall, this fast, easy, and accurate method can facilitate the direct identification and antibiotic susceptibility testing of bacteria in positive blood cultures.

A Simplified Sample Preparation Method for the Assessment of Plasma Ascorbic Acid in Clinical Settings.

Vitamin C deficiency is difficult to diagnose on the basis of clinical presentation alone and requires plasma levels for confirmation. Reference laboratories typically specify shipment of plasma on dry ice. This requirement may complicate clinic work flow and delay vitamin C measurement. Additionally, patients with vitamin C deficiency may experience unnecessary testing and increased health-care costs, as other diagnoses are often considered first. We examined an alternative, more practical shipping method. Plasma was collected from 17 healthy volunteers by use of heparin tubes with gel separators, and all tubes were centrifuged immediately to separate the plasma layer from the cells. Baseline vitamin C was measured in plasma obtained immediately after specimen collection. Remaining sample tubes were held in Styrofoam containers with cold packs for 30 h or 48 h, followed by vitamin C measurement. Additional samples were exposed to conditions that simulated harsher shipping conditions. Mean plasma vitamin C was 69.6 μmol/L (SD = 21.5 μmol/L). Vitamin C losses were 5.4% at 30 h (SD = 5.55%, P < 0.05) and 7.6% at 48 h (SD = 5.56%, P < 0.05), which is slightly more than freeze-and-thaw treatment (average loss of 1.4%, SD = 6.9%, NS). The vitamin C method had an intraday variation of 1.88%. Vigorous shaking of 2 samples for 24 h resulted in a -1.9% change in 1 sample, and a +4.1% change in another sample. Exposure of the shipping container to elevated temperature (35 °C for 30 h) did not change the internal temperature of the container. The shipping procedure uses routine sample handling, standard vacutainers, and can be replicated by health-care centers seeking to evaluate patient vitamin C status.

A Simple and Highly Sensitive Quantitation of Eicosanoids in Biological Samples Using Nano-flow Liquid Chromatography/Mass Spectrometry.

A simple sample preparation method for eicosanoid was developed by the combination of deproteinization and nanoLC-ESI-MS/MS. Eicosanoids are a group of bioactive lipid mediators, present in trace amounts in the body. Therefore, an analytical method for eicosanoids requires superior sensitivity. The method described in this report, which takes advantage of the highly sensitive power of nanoLC-ESI-MS/MS, enabled a simplification of the sample-preparation process. Eicosanoid extraction was performed just by homogenization in methanol with subsequent phospholipid removal, and then the liquid phase was directly subjected to nanoLC-ESI-MS/MS analysis without a condensation process. The quantitation range achieved 0.01 - 100 ng/mL for thromboxane B2, and 0.05 - 100 ng/mL for prostaglandin E2, prostaglandin D2, prostaglandin F2, leukotriene B4, 6-keto prostaglandin F1α and 11-dehydro thromboxane B2. Rat brain sample analyses demonstrated the feasibility of the quantification of those seven eicosanoids from biological samples.

A simple method for chromium speciation analysis in contaminated water using APDC and a pre-heated glass tube followed by HPLC-PDA

In this study, a simple sample preparation method was developed for the determination of tri-and hexavalent chromium in water samples. It utilizes a pre-heated customized glass tube (CGT), to supply the heat energy required for the reaction of Cr(III) with ammonium pyrrolidinedithiocarbamate (APDC). The products of the Cr complexes, tris(1-pyrrolidinecarbodithioato)chromium(III) and bis(1-pyrrolidinecarbodithioato)[1-pyrrolidinecarbodithio(thioperoxoato)]chromium(III) were chromatographed with Shimadzu LC-20AT and Zobax Eclipse C18 (150mm × 4.6mm, 5µm) column using ACN: Water, (7:3, v/v) as the mobile phase. The concentration of Cr(III) ranged from 0.06mgL−1 to 0.09mgL−1 and that of Cr(VI) was between 0.02mgL−1 to 0.04mgL−1 in the samples. Percentage recoveries from spiked real samples were between 87% (tap water) to 110% (wastewater) for Cr(III) and 92% (pond water) to 117% (tap water) for Cr(VI). The limits of detection (LODs) were 0.0029mgL−1 and 0.0014mg/L−1 for Cr(III) Cr(VI) respectively. While the limits of quantitation (LOQs), were 0.0098mgL−1 and 0.0047mgL−1 for Cr(III) and Cr(VI) respectively. Method precision (RSD (%)) was 3.3% and 3.5% for Cr(III) and Cr(VI) respectively. The developed method was applied for the speciation analysis of chromium in drinking water, tap water, wastewater, river water, and pond water samples. Our findings proved the method is simple and inexpensive. The method was validated by the analysis of a certified reference material (CRM) SLRS-4. The percentage recovery and RSD(%) from the spiked CRM were 91% and 115% and 0.32% and 1.4% for Cr(III) and Cr(VI) respectively.

Simultaneous determination of thirteen different steroid hormones using micro UHPLC-MS/MS with on-line SPE system

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LOQ). Micro UHPLC coupled to sensitive tandem mass spectrometry provides state of the art solution for such analytical problems. Using on-line SPE with column switching on a micro UHPLC–MS/MS system allowed to decrease LOQ without any complex sample preparation protocol. The presented method is capable of reaching satisfactory low LOQ values for analysis of thirteen different steroid molecules from human plasma without the most commonly used off-line SPE or compound derivatization. Steroids were determined by using two simple sample preparation methods, based on lower and higher plasma steroid concentrations. In the first method, higher analyte concentrations were directly determined after protein precipitation with methanol. The organic phase obtained from the precipitation was diluted with water and directly injected into the LC–MS system. In the second method, low steroid levels were determined by concentrating the organic phase after steroid extraction. In this case, analytes were extracted with ethyl acetate and reconstituted in 90/10 water/acetonitrile following evaporation to dryness. This step provided much lower LOQs, outperforming previously published values. The method has been validated and subsequently applied to clinical laboratory measurement.

Determination of total phthalate in cosmetics using a simple three-phase sample preparation method.

A simple sample preparation method requiring minimal organic solvents is proposed for the determination of the total phthalate content in cosmetics by high-performance liquid chromatography-tandem mass spectrometry. The hydrolysis of phthalates and purification of interfering substances were performed in a three-phase system that included an upper n-hexane phase, a middle ethanol phase, and a lower aqueous alkali solution. This three-phase system utilized an incremental purification strategy. The apolar ingredients were extracted with n-hexane, the polar pigments accumulated in the ethanol phase, and the hydrolysis product, phthalic acid, remained in the hydrolysate. Under the optimized conditions, the correlation coefficients (r) for the calibration curves were 0.998-0.999 in the range 0.60-12molL-1. The limit of detection was 5.1μmolkg-1, and the limit of quantification was 9.2μmolkg-1. The recoveries varied from 84 to 97% with RSDs equal to or lower than 11%. The intra-day and inter-day repeatability values, expressed as the relative standard deviation, were less than 8.7 and 9.8, respectively. No obvious matrix effect existed in the different cosmetics matrices. The validated method was applied for the analysis of 57 commercial cosmetic samples. Graphical abstract Analysis of phthalates in cosmetics using a three-phase preparation method.

Application of the Ultrasonic-Assisted Extraction and Dispersive Liquid–Liquid Microextraction for the Analysis of AFB1 in Egg

A fast and simple sample preparation method based on dispersive liquid–liquid microextraction (DLLME) and ultrasonic-assisted extraction (UAE) was proposed in order to determine aflatoxin B1 (AFB1) in eggs. In this study, AFB1 was first extracted from matrixes of egg samples with acetonitrile/water 80:20 (V/V), then its eluents were utilized as dispersant of the followed UAE-DLLME for further purification and concentration of AFB1. All parameters that could affect the effectiveness of this method were investigated and optimized such as types and volumes of extractant and dispersive solvents, volume of water, pH of extracted solution, salt addition, ultrasonic time, ultrasonic temperature, and centrifugation time. Quantification method was HPLC-UV which does not require AFB1 derivatization in samples. Under the optimum UAE-DLLME conditions (240 μL CHCl3 as extraction solvent, 1.2 mL acetonitrile 80% extract as disperser solvent, water volume 3 mL, sample pH 5.6, ultrasonic time 2, and ultrasonic temperature 35 °C) recovered result were between 91 and 94%. The limits of detection and quantification were 0.12 and 0.32 μg kg−1, respectively. Real samples were analyzed by the proposed UAE-DLLME method and then compared with confirmative immunoaffinity column chromatography (IAC) clean-up. The proposed UAE-DLLME method is economical, simple, high enrichment factor, quick, and environmental friendly.

Toward a Noninvasive, Label-Free Screening Method for Determining Spore Inoculum Quality of Penicillium chrysogenum Using Raman Spectroscopy.

We report on a label-free, noninvasive method for determination of spore inoculum quality of Penicillium chrysogenum prior to cultivation/germination. Raman microspectroscopy providing direct, molecule-specific information was used to extract information on the viability state of spores sampled directly from the spore inoculum. Based on the recorded Raman spectra, a supervised classification method was established for classification between living and dead spores and thus determining spore inoculum quality for optimized process control. A fast and simple sample preparation method consisting of one single dilution step was employed to eliminate interfering signals from the matrix and to achieve isolation of single spores on the sample carrier (CaF2). Aiming to avoid any influence of the killing procedure in the Raman spectrum of the spore, spores were considered naturally dead after more than one year of storage time. Fluorescence staining was used as reference method. A partial least squares discriminant analysis classifier was trained with Raman spectra of 258 living and dead spores (178 spectra for calibration, 80 spectra for validation). The classifier showed good performance when being applied to a 1 µL droplet taken from a 1:1 mixture of living and dead spores. Of 135 recorded spectra, 51% were assigned to living spores while 49% were identified as dead spores by the classifier. The results obtained in this work are a fundamental step towards developing an automated, label-free, and noninvasive screening method for assessing spore inoculum quality.