Sample Vial Research Articles

On-line coupling of two-phase microelectroextraction to capillary electrophoresis – Mass spectrometry for metabolomics analyses

We have integrated two-phase electroextraction (EE), capillary electrophoresis (CE) and mass spectrometry (MS) to combine rapid sample extraction, high performance separation and sensitive detection of metabolites. EE took place from a sample vial containing organic donor phase into a ~100 nL droplet of background electrolyte, which was hanging at the inlet of the capillary. In order to enable the EE process while the outlet of the capillary was connected to the MS several modifications were made. A modification was made to the sheath-liquid CE-MS interface, based on the use of a corrosion-resistant titanium ESI sprayer and a 6-port valve to switch between the CE-MS sheath liquid and the EE make-up liquid. Furthermore, a counter-pressure was applied, in order to prevent EOF from retracting the droplet during EE.Then, using five model metabolites (namely, leucine, isoleucine, adenosine, phenylalanine and guanosine) and crystal violet (CV) the extraction time and voltage were optimized and found to be 2000 s and 1 kV, respectively. Using these optimized conditions, the effect of various sodium chloride concentrations was examined to assess the influence of varying salt concentrations in biological samples. A set of 9 amino acids was used to validate the method. The detection limits ranged between 5 and 100 nM. LODs were improved 50–250 times in comparison with conventional CE-MS. Finally, to demonstrate the potential of the EE-CE-MS platform for bioanalysis of volume-limited samples, a urine sample of 300 nL was analyzed. This resulted in detection of 122 putative metabolites. The results indicate that EE-CE-MS could become a powerful tool for metabolomics analyses of volume-limited samples.

Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology.

We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30–39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.

Deaths related to nitrogen inhalation: Analytical challenges

Dinitrogen (N2) has been increasingly connected to suicidal deaths. The analysis of N2 in post-mortem cases still represents a major challenge in forensic toxicology and circumstantial data has so far played a major role for the determination of the cause of death. In this paper, after presenting a review of cases of N2 intoxication described in forensic literature, we report the application of two approaches in order to quantify an excess of N2 in post-mortem whole blood collected from a case of suicide by nitrogen inhalation.N2 analyses were performed by GC–MS on the suicidal case and on controls taken from 10 autopsy cases with similar PMI (5 traumatic deaths and 5 deaths by asphyxia). The percentage of N2 was estimated by building a five-point N2 peak area calibration curve (0, 15.6 %, 62.4 % 78.1 %, 100 %) and through an external QC, assessing linearity, accuracy and precision, LLOQ, specificity and stability of N2 in the sample vial. Percentage of N2 of the case was significantly higher than the post-mortem controls (p<0.05). The N2/O2 ratio of the case and controls was also calculated as an additional indicator, and was significantly higher in the case (p<0.05). The strengths and the limitation of both methods are reported in the paper.Toxicological confirmation for N2 are rarely performed when the cause of death is evident, probably due to the lack of validated methods and the complexity of the interpretation of N2 concentration in biological fluids. The presented methods can be rapidly and profitably applied with instrumentation normally available in forensic laboratories.

Improved intact peptide and protein quantitation by LC-MS: Battling the deleterious effects of analyte adsorption.

Peptide and protein quantitation by liquid chromatography-mass spectrometry relies on the assumption of linear signal response with concentration. At low concentrations, analyte adsorption to pipette tips, sample vials and equipment can have significant deleterious effects on signal response. Meanwhile at high concentrations, linearity breaks down due to competitive ionization, signal suppression, and the formation of peptide or protein multimers. These effects result in calibration curves that are more sigmoidal than linear. Linearity at low protein levels for identification and quantitation is of paramount importance in the discovery and validation of biomarker molecules. Herein, we demonstrate the benefits of using commercial low-bind microcentrifuge tubes and LC vials on the response of a 27-mer peptide, Vn96, and the intact proteins apomyoglobin and carbonic anhydrase. Linear curves were acquired for Vn96 while apomyoglobin required the addition of intact carbonic anhydrase as an adsorption competitor to achieve linearity. A linear calibration curve for carbonic anhydrase was also acquired by using the polypeptide ubiquitin as an adsorption competitor and internal standard. Linear response was recorded for approximately two orders of magnitude for apomyoglobin and carbonic anhydrase and three orders of magnitude for Vn96 with detection limits ranging from 0.33 to 19 fmol/µL. Finally, we used low-bind vials for the online enzymatic digestion of apomyoglobin where a high concentration of apomyoglobin acted as an adsorption blocker for the low level trypsin enzyme. Fortunately, the liberated tryptic peptides showed no affinity for the walls of the low-bind vials. In this study, we take a comprehensive approach to combat analyte adsorption by showing the significance of utilizing low-bind vials and adsorption competitors to greatly improve upon signal sensitivity at low concentrations of target molecules. The use of these methodologies should improve the low-level detection of molecules by mass spectrometry.

Impact of ion suppression by sample cap liners in lipidomics

Contamination from the polymeric material released by vial caps used for sample introduction in liquid chromatography can significantly affect the signal of the analyte of interest. In particular, repeated injections from the same sample vial can enhance this suppressing effect. Multiple injections of the same sample are often used in metabolomics and lipidomics during routine analyses. Here we demonstrate how the presence of contaminant polymers, originating from the vial closures, significantly influences the estimation of the relative amount of endogenous lipids in human plasma. Furthermore, this can negatively impact other operations in mass spectrometric analysis, such as instrument equilibration and tuning or the common use of technical replicates to improve confidence in data interpretation. Our observations provide critical information on how to improve future analyses through the use of appropriate vial caps, solvents, chromatographic separations and equipment.

High-precision stable isotope analysis of

Oxygen (δ18 O) and carbon (δ13 C) isotope analysis of foraminifera and other CaCO3 samples has been a key technique for paleoceanographical and paleoclimatological research for more than 60 years. There is ongoing demand for the analysis of ever smaller CaCO3 samples, driven, for example, by the desire to analyse single specimen planktic foraminifera, or small samples of tooth enamel. We present a continuous-flow mass spectrometric technique that uses cryo-focusing of sample CO2 to analyse CaCO3 samples in a weight range between 10 and 3 μg. These are considerably lower sample weights than achievable on most currently available standard instrumentation. The technique is automated, so that sample throughput lies at >60 samples per day. The method involves an on-line vial-flushing routine designed to remove machine drift due to blank CO2 build-up in the sample vials. In a series of experiments the effect of blank CO2 build-up is quantified, and outgassing from the chlorobutyl septa identified as the source. An improved flushing routine together with the use of a cryo-focusing step in the analysis is demonstrated to provide the analytical stability and sensitivity to analyse CaCO3 samples in a weight range between 10 and 3 μg at ≤0.1‰ precision (1σ) for both δ18 O and δ13 C values. The technique yields similarly precise results for the analysis of the structural carbonate fraction of small tooth enamel samples. This study demonstrates that high-precision oxygen and carbon isotope analysis is possible on CaCO3 samples smaller than 5 μg by use of a continuous-flow isotope technique. Of key importance are (1) the application of a cold trap that drastically reduces sample gas loss, and (2) a modified flushing regime that eliminates increasing background CO2 build-up in sample vials during longer automated sample runs.

Accelerating gene function discovery by rapid phenotyping of fatty acid composition and oil content of single transgenic T1 Arabidopsis and camelina seeds.

Arabidopsis is wildly used as a model plant and camelina is increasingly used for oilseed research and applications. Although the Arabidopsis genome has been sequenced for two decades, the functions of many lipid‐related genes and their regulators have not been well characterized. Improvements in the efficiency and accuracy of gene investigations are key to effective discovery of gene function and downstream bioengineering of plant oil quantity and quality. In this study, a visible marker was used to quickly identify transgenic T1 seeds and a method has been developed to phenotype fatty acid compositions and oil content of single T1 seeds. A whole seed direct transmethylation method was first optimized with multiple seeds and incubation at 85°C for 2 hours in a transmethylation solvent (5% H2SO4 in methanol with 30% toluene cosolvent) is recommended. Based on this method, a single Arabidopsis seed mini‐transmethylation (SAST) method has been established in a 1.5 ml GC sample vial with 200 μl transmethylation solvent. Characteristics of the method were evaluated and it was used to phenotype transgenic T1 seeds expressing AtFAD2 or RcWRI1. Our results indicate that fatty acid composition of T1 individual seeds are consistent with those of pools of multiple seeds from higher generations. However, oil content per individual seed varied substantially and therefore pooling five seeds is recommended for phenotyping oil content of T1 seeds. Additionally, a whole camelina single‐seed direct transmethylation was evaluated and results confirm its feasibility. The suitability of partial seed analysis of camelina was investigated but variation in composition of different seed tissues limits this approach.

Investigating the oxidation state of Fe from LiFePO4 -based lithium ion battery cathodes via capillary electrophoresis.

A capillary electrophoresis (CE) method with ultraviolet/visible (UV-Vis) spectroscopy for iron speciation in lithium ion battery (LIB) electrolytes was developed. The complexation of Fe2+ with 1,10-phenantroline (o-phen) and of Fe3+ with ethylenediamine tetraacetic acid (EDTA) revealed effective stabilization of both iron species during sample preparation and CE measurements. For the investigation of small electrolyte volumes from LIB cells, a sample buffer with optimal sample pH was developed to inhibit precipitation of Fe3+ during complexation of Fe2+ with o-phen. However, the presence of the conducting salt lithium hexafluorophosphate (LiPF6 ) in the electrolyte led to the precipitation of the complex [Fe(o-phen)3 ](PF6 )2 . Addition of acetonitrile (ACN) to the sample successfully re-dissolved this Fe2+ -complex to retain the quantification of both species. Further optimization of the method successfully prevented the oxidation of dissolved Fe2+ with ambient oxygen during sample preparation, by previously stabilizing the sample with HCl or by working under counterflow of argon. Following dissolution experiments with the positive electrode material LiFePO4 (LFP) in LIB electrolytes under dry room conditions at 20°C and 60°C mainly revealed iron dissolution at elevated temperatures due to the formation of acidic electrolyte decomposition products. Despite the primary oxidation state of iron in LFP of +2, both iron species were detected in the electrolytes that derive from oxidation of dissolved Fe2+ by remaining molecular oxygen in the sample vials during the dissolution experiments.

Um acervo centenário: a história da Coleção de Formigas (Insecta: Hymenoptera: Formicidae) do Museu de Zoologia da Universidade de São Paulo

A Coleção de Formigas do Museu de Zoologia da Universidade de São Paulo (MZSP) representa uma amostra inestimável da biodiversidade de formigas, principalmente da região neotropical. Seu acervo foi formado a partir da contribuição de naturalistas-viajantes, de mirmecólogos reconhecidos por suas contribuições científicas, do desenvolvimento de pesquisas e por sua atuação como instituição fiel depositária. Abriga uma expressiva quantidade de material-tipo, comporta 4.741 espécimes-tipos de 892 espécies, amplamente distribuídos. A prática curatorial constante e adequada é um dos grandes desafios da coleção, que serve como base para a formação de pessoal especializado em diversas áreas integrantes do grande campo de biodiversidade. No presente artigo, apresentamos um breve histórico do MZSP e um relato mais detalhado sobre a criação, a expansão e a qualificação do acervo da Coleção de Formigas do MZSP, que atualmente conta com quase 440 mil espécimes em via seca, além de amostras não quantificadas em via úmida.

In vivo solid-phase microextraction swab sampling of environmental pollutants and drugs in human body for nano-electrospray ionization mass spectrometry analysis

In vivo sampling and sensitive detection of environmental pollutants and drugs in human body play a crucial role in understanding human health. In this study, in vivo solid-phase microextraction (SPME) swab was fabricated using a SPME fiber and a medical cotton swab for noninvasive sampling and extraction of environmental pollutants and drugs in human oral cavity, nasal cavity and on skin surface. After sampling, SPME was coupled with nano-electrospray ionization mass spectrometry (nanoESI-MS) for desorption, ionization, and detection of the extracted analytes. As a result, limit of detection (LOD) and limit of quantification (LOQ) of nicotine in oral fluid were found to be 1.0 pg/mL (S/N ≥ 3) and 4.0 pg/mL (S/N ≥ 10), respectively. Linear dynamic signal responses of nicotine exhibited excellent linearity (R2 = 0.9996) in human oral fluid ranging from 0.1 to 50 ng/mL. The coefficient of variation (CV) values of SPME swab for five measurements from sample vials and human body were 5.1–6.7% and 22.7–32.6%, respectively. Rapid analysis of a single sample could be completed within 10 min. Overall, our results demonstrated that SPME swab-MS is a promising noninvasive method for enhanced detection of analytes in human body.

Molecular identification of Neoechinorhynchus rutili parasite diagnosed in some fish species caught in Menzelet dam lake in Kahramanmaras province (Turkey)

This study details the molecular identification of the parasite Neoechinorhynchus rutili diagnosed in fish (Capoeta barroisi, Cyprinus carpio, Barbus rajanorum) caught in Menzelet Dam Lake in Kahramanmaras Province, Turkey. Parasite samples were obtained from the intestines of fish caught from January to June of 2013. The collected parasites were stored in sample vials containing 70% alcohol. Using staining methods and based on morphology, 120 N. rutili specimens were identified. DNA isolation of N. rutili was accomplished using special tissue sets for parasites. Specific primers were utilized in the molecular identification of N. rutili using polymerase chain reaction (PCR) and it was possible to verify that all of the parasites contained N. rutili molecules. In conclusion, using multiple methods we successfully identified and confirmed the presence of N. rutili parasites in the fish caught in Kahramanmaras Province. The process of identification of N. rutili using morphology and staining methods is time-consuming; however, PCR was successfully performed in a short time to accomplish the same results. The success of this study may lead to more original and extensive work aimed at the efficient molecular identification of parasitic agents found in fish.

On-line recovery system coupled to a Rock-Eval® device: An analytical methodology for characterization of liquid and solid samples

The Rock-Eval® device has been widely used to identify the type and the thermal maturity of sedimentary organic matter, as well as for quantifying the total organic carbon content. Traditionally, it is a screening tool to estimate the petroleum generation potential of source rocks using standardized parameters. More recently, a new Rock-Eval® method (Shale Play™) was proposed for the investigation of the hydrocarbon content of liquid-rich tight rock samples. In this study, we describe a dual vacuum and on-line system that was developed to recover most compounds that are thermally released during a Rock-Eval® Shale Play™ analysis. Thermally vaporized products are divided so that half is analyzed by the Rock-Eval® flame ionization detector (FID) while the other portion is cryogenically trapped in the on-line recovery system. The trapped products can then be transferred via a vacuum line system into a sample vial for subsequent molecular and/or isotopic composition analyses. The recovery vacuum line volumes were calibrated using known quantities of gas (CH4 and CO2). Sample transfer without isotopic fractionation was demonstrated for CO2 evolved from Rock-Eval® preparation of pure carbonate standards (siderite, magnesite and azurite). Recovery efficiencies were first measured on C8-C16n-alkane standards and then on produced oil samples. Results indicate a high quantitative recovery and an accurate mass balance of most compounds released during the Shale Play™ Sh0 thermovaporization step (100–200 °C). Thermally vaporized compounds released at higher temperatures Sh1 (200–350 °C) are recovered at lower efficiencies, but are still suitable for subsequent characterization. The coupled Rock-Eval® and recovery system could have applications beyond petroleum geochemistry.

Pyrolyzed cotton balls for protein removal: Analysis of pharmaceuticals in serum by capillary electrophoresis

Because of the inherent affinity of proteins for bare, fused silica capillaries, the analysis of protein-containing samples has proven a challenging task for capillary electrophoresis. The adsorption of proteins to the capillary walls effectively changes the zeta potential and thus affects the electro-osmotic flow leading to significant shifts in migration time, peak broadening, and poor reproducibility. While there are several well-known methods to remove proteins from samples prior to the analysis (including precipitation) or to prevent their adsorption to the capillary (semi-permanent coatings), those approaches are often expensive, time consuming, or simply unreliable. Aiming to address these needs, this manuscript reports on the use of pyrolyzed cotton balls, as a simple and widely accessible hydrophobic material to remove proteins from serum samples. The material retains enough flexibility so it can be placed directly into the sample vials and has enough capacity to capture more than 75% of the proteins in the sample (1% dilution of 1 mL of serum). The advantages of the material are demonstrated by performing the analysis of five representative drugs (in serum) by capillary electrophoresis obtaining a change in migration time of only 5 ± 1%, after 10 consecutive runs.

Comparison of multiple calibration approaches for the determination of volatile organic compounds in air samples by solid phase microextraction Arrow and in-tube extraction

Several calibration approaches were evaluated for the quantitation of volatile organic compounds in air using miniaturized exhaustive and non-exhaustive sampling techniques, such as in-tube extraction (ITEX) and solid phase microextraction (SPME) Arrow. Eleven compounds, 2-ethyl-hexanol, hexanal, nonanal, toluene, ethyl-benzene, methyl isobutyl ketone, acetophenone, p-cymene, α-pinene, trimethylamine and triethylamine, all them found in the natural air samples, were selected as model analytes. Liquid injection, liquid standard addition to the sorbent bed and gas phase standards provided by an automatic permeation system, were evaluated in the case of ITEX packed with laboratory-made 10% polyacrylonitrile (PAN) material. Two different approaches, based on sampling of gas phase compounds from the permeation system and from sample vial containing gas phase standards, were evaluated for SPME Arrow with two different coatings, commercial divinylbenzene-poly(dimethylsiloxane) (DVB-PDMS) and laboratory-made mesoporous Mobil Composition of Matter No. 41 (MCM-41). In addition, interface model approach was used for the calculation of the real concentration of the target analytes in the sample from the total amount of analytes injected into the GC–MS in the case of SPME Arrow. Similar results were obtained with the different approaches used for the quantitation by ITEX and SPME Arrow. However, the use of gas phase standards with sample matrix similar to the natural samples, allowed the permeation system to provide the most reliable results for the quantitation of the target analytes. For this approach, linearity (expressed as r2 values) ranged between 0.991 and 0.999. The limit of detection ranged from 0.5 µg/m3 (trimethylamine, MCM-41) to 2.2 × 10−4 µg/m3 (methyl isobutyl ketone, MCM-41). In addition, the use of the fully automated permeation system provided good reproducibility values that were between 1.4% (acetophenone, MCM-41) and 7.8% (methyl isobutyl ketone, 10% PAN). The linear ranges were at least 3 order of magnitude for all the studied analytes with the exception of the calibration curve developed for trimethylamine with SPME Arrow (linear ranges between LOQ and 4.9 µg/m3 (DVB-PDMS) and LOQ and 9.8 µg/m3 (MCM-41)).

Sensitive determination of methylmercury δ13C compound specific stable isotopic analysis by purge and trap gas chromatography combustion isotope ratio mass spectrometry

Despite several decades of mercury research, answering fundamental questions on where and how methylmercury (CH3Hg) toxin is naturally produced in aquatic ecosystems, is still highly challenging. Investigating complex and/or coupled processes in the context of global changes requires new high-resolution analytical tools. The purpose of the compound specific carbon stable isotopic analysis (δ13C-CSIA) of the methyl group of methylmercury (CH3Hg), is to explore how the carbon cycle contributes to CH3Hg sources and formation pathways. The main problem associated with recent CH3Hg δ13C-CSIA methods is the limited sensitivity when using Liquid Injection (LI)-GC-C-IRMS techniques, requiring several micrograms of CH3Hg (as Hg). In this work, we present the development and application of an original Purge-&-Trap system (PT) coupled to a GC-C-IRMS with the purpose of transferring and analyzing the total amount of CH3Hg available in a sample vial in the low nanogram range. The new PT-GC-C-IRMS system enhance the sensitivity by a factor better than 200, relative to LI-GC-C-IRMS, by minimizing the sample mass requirements. The δ13CCH3Hg values obtained, following the same sample derivatization approach coupled to PT-GC-C-IRMS (−53.5 ± 1.9 ‰), were in good agreement with the ones obtained in a previous study (−53.8 ± 1.1 ‰). The standard solution was prepared from the same salt, requesting only 25–200 ng of CH3Hg (as Hg). This new methodology represents a milestone towards the analysis of large array of biological samples displaying CH3Hg concentrations in the low-mid ng g−1 range, in order to explore the meaning of the carbon stable isotopic signature of CH3Hg in the environment.

Efficient Anti-solvent-free Spin-Coated and Printed Sn-Perovskite Solar Cells with Crystal-Based Precursor Solutions

Summary Tin-based perovskite solar cells (PSCs), with more consummate optical band gaps, lower exciton-binding energies, and higher charge-carrier mobility, have not attracted tremendous research efforts compared with the lead-based ones that have a record power conversion efficiency (PCE) of 24.2%. The major challenges for Sn-based research are significantly low open-circuit voltage, poor reproducibility, and environmental instability. It has been identified that the oxidation processes take place during the preparation of precursor solutions and fabrication of films, due to the low redox potential of the stannous absorption layer. Herein, we propose two strategies of crystal-resolving and recrystallization technology to reduce the oxidation process. As a result, PCEs of 8.9% (active area of 0.04 cm2) and 5.5% (active area = 1.01 cm2) are attained using the inverted p-i-n structure, which will lead to a revival of lead-free PSC research.

Direct capillary electrophoresis analysis of basic and acidic drugs from microliter volume of human body fluids after liquid-phase microextraction through nano-fibrous membrane.

In the present work, a disposable microextraction device with a polyamide 6 nano-fibrous supported liquid membrane (SLM) is employed for the pretreatment of minute volumes of biological fluids. The device is placed in a sample vial for an at-line coupling to a commercial capillary electrophoresis instrument with UV-Vis detection (CE-UV) and injections are performed fully automatically from the free acceptor solution above the SLM with no contact between the capillary and the membrane. Up to 4-fold enrichment of model basic (nortriptyline, haloperidol, loperamide, and papaverine) and acidic (ibuprofen, naproxen, ketoprofen, and diclofenac) drugs is achieved by optimizing the ratio of the donor to the acceptor solution volumes (16 to 4 μL, respectively). The actual setup enables SLM extractions from less than a drop of sample and is suitable for pretreatment of scarce human body fluids. Two unique methods are reported for efficient clean-up and enrichment of the basic and acidic drugs from capillary blood (formed as dried blood spot), serum, and urine samples, which enable their determination at therapeutic and/or toxic levels. The hyphenation of the SLM extraction with CE-UV analysis provides good repeatability (RSD, 2.4-14.9%), linearity (r2, 0.988-1.000), sensitivity (LOD, 0.017-0.22 mg L-1), and extraction recovery (ER, 20-106%) at short extraction times (10 min) and with minimum consumption of samples and reagents. Graphical abstract.

Determination of Dithiocarbamate Mancozeb Residues in Milk Samples Using GC-MS Method

The present study was performed to validate gas chromatography mass spectrometry analytical method for the determination of mancozeb residues in milk samples. The analytical method is based on the extraction procedure by using mineral acids. The extraction solution was sucked into the flask, the dripping funnel was quickly exchanged for the gas inlet. After 2 hours, the adsorption tube containing the isooctane was removed and closed. The solution was transferred into a sample vial and analysed using gas chromatography- mass spectroscopy (GC-MS). A Rxi-624Sil MS (30 m length x 0.32mm I.D. x 1.8 μm particle size capillary column is used for the separation. The method has linearity over the range 0.05 to 10.0 μg/mL. Recovery study was conducted at 0.05 and 0.5 mg/kg fortification levels. The average mean recoveries were calculated as 86.23 % at 0.05 mg/kg level and 92.69 % at 0.5 mg/kg level. The limit of quantification (LOQ) in milk was established as 0.05 mg/kg.

Rapid one-step enzyme immunoassay and lateral flow immunochromatographic assay for colistin in animal feed and food

BackgroundColistin (polymyxin E) is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention, treatment, and growth promotion. However, the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings, and its residue in animal-origin food may also pose serious health hazards to humans. Thus, it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food.ResultsA one-step enzyme-linked immunosorbent assay (ELISA) and a lateral flow immunochromatographic assay (LFIA) for colistin were developed based on a newly developed monoclonal antibody. The ELISA showed a 50% inhibition value (IC50) of 9.7 ng/mL with assay time less than 60 min, while the LFIA had a strip reader-based detection limit of 0.87 ng/mL in phosphate buffer with assay time less than 15 min. For reducing the non-specific adsorption of colistin onto sample vial, the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy. The spiked recovery experiment showed that the recoveries of colistin from feed, milk and meat samples were in the range of 77.83% to 113.38% with coefficient of variations less than 13% by ELISA analysis and less than 18% by LFIA analysis, respectively. Furthermore, actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis.ConclusionsThe developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.

Improved Nanoflow RPLC-CZE-MS/MS System with High Peak Capacity and Sensitivity for Nanogram Bottom-up Proteomics.

Novel mass spectrometry (MS)-based proteomic tools with extremely high sensitivity and high peak capacity are required for comprehensive characterization of protein molecules in mass-limited samples. We reported a nanoRPLC-CZE-MS/MS system for deep bottom-up proteomics of low micrograms of human cell samples in previous work. In this work, we improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically via employing bovine serum albumin (BSA)-treated sample vials, improving the nanoRPLC fraction collection procedure, and using a short capillary for fast CZE separation. The improved nanoRPLC-CZE produced a peak capacity of 8500 for peptide separation. The improved system identified 6500 proteins from a MCF7 proteome digest starting with only 500 ng of peptides using a Q-Exactive HF mass spectrometer. The system produced a comparable number of protein identifications (IDs) to our previous system and the two-dimensional (2D) nanoRPLC-MS/MS system developed by Mann's group with 10-fold and 4-fold less sample consumption, respectively. We coupled the single-spot solid phase sample preparation (SP3) method to the improved nanoRPLC-CZE-MS/MS for bottom-up proteomics of 5000 HEK293T cells, resulting in 3689 protein IDs with the consumption of a peptide amount that corresponded to only roughly 1000 cells.