Portable loop-mediated isothermal amplification device with spectrometric detection for rapid pathogen identification.

With the rise in extreme weather due to global warming, coupled with globalization facilitating the spread of infectious diseases, there's a pressing need for portable testing platforms offering simplicity, low cost, and remote transmission, particularly beneficial in resource-limited and non-urban areas. We have developed a portable device using loop-mediated isothermal amplification (LAMP) with spectrometric detection to identify Salmonella Typhimurium DNA. The device utilizes the LinkIt 7697 microcontroller and a microspectrometer to capture and transmit spectral signals in real-time, allowing for improved monitoring and analysis of the reaction progress. We built a hand-held box containing a microspectrometer, thermoelectric cooler, ultraviolet LED, disposable reaction tube, and homemade thermal module, all powered by rechargeable batteries. Additionally, we conducted thorough experiments to ensure temperature accuracy within 1 °C under thermal control, developed a heating module with a LinkIt 7697 IoT development board to heat the DNA mixture to the reaction temperature within 3 min, and integrated foam insulation and a 3D-printed frame to enhance the device's thermal stability. We successfully demonstrated the amplification of Salmonella Typhimurium DNA with an impressive sensitivity of 2.83 × 10-4 ng/µL. A remote webpage interface allows for monitoring the temperature and fluorescence during the LAMP process, improving usability. This portable LAMP device with real-time detection offers a cost-effective solution for detecting Salmonella Typhimurium in food products. Its unique design and capabilities make it a promising tool for ensuring food safety.

Development and validation of a quantitative wipe sampling method to determine platinum contamination from antineoplastic drugs on surfaces in workplaces at Swedish hospitals.

INTRODUCTION: Antineoplastic drugs (ADs) are frequently used pharmaceuticals in the healthcare, and healthcare workers can be occupationally exposed to ADs. Monitoring of surface contamination is a common way to assess occupational exposure to ADs. The objective was to develop and validate a sensitive and quantitative monitoring method to determine surface contaminations of Pt as a marker for Pt-containing ADs. The surface contaminations of Pt-containing ADs were monitored at four Swedish hospital workplaces. METHODS: An analytical method was developed based on inductively coupled plasma mass spectrometry. The wipe sampling procedure was validated regarding different surface materials. The stability of collected wipe samples was investigated. Workplace surfaces were monitored by wipe sampling to determine contaminations of Pt-containing ADs. RESULTS: A wipe sampling and analytical method with a limit of detection of 0.1 pg Pt/cm2 was developed. Pt was detected in 67% of the wipe samples collected from four workplaces, and the concentrations ranged from <0.10 to 21100 pg/cm2. In 4% of samples, the detected surface contaminations of Pt in three hospital wards were above proposed hygienic guidance value (HGV) of Pt. In the hospital pharmacy, 9% of the detected surface contaminations of Pt were above lowest proposed HGV. CONCLUSIONS: A user-friendly, specific, and sensitive method for determination of surface contaminations of Pt from ADs in work environments was developed and validated. A large variation of contaminations was observed between detected surface contaminations of Pt in samples collected in wards, and it likely reflects differences in amounts handled and work practices between the wards.

The "depict" strategy for discovering new compounds in complex matrices: Lycibarbarspermidines as a case.

The comprehensive detection and identification of active ingredients in complex matrices is a crucial challenge. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is the most prominent analytical platform for the exploration of novel active compounds from complex matrices. However, the LC-HRMS-based analysis workflow suffers from several bottleneck issues, such as trace content of target compounds, limited acquisition for fragment information, and uncertainty in interpreting relevant MS2 spectra. Lycibarbarspermidines are vital antioxidant active ingredients in Lycii Fructus, while the reported structures are merely focused on dicaffeoylspermidines due to their low content. To comprehensively detect the new structures of lycibarbarspermidine derivatives, a "depict" strategy was developed in this study. First, potential new lycibarbarspermidine derivatives were designed according to the biosynthetic pathway, and a comprehensive database was established, which enlarged the coverage of lycibarbarspermidine derivatives. Second, the polarity-oriented sample preparation of potential new compounds increased the concentration of the target compounds. Third, the construction of the molecular network based on the fragmentation pathway of lycibarbarspermidine derivatives broadened the comprehensiveness of identification. Finally, the weak response signals were captured by data-dependent scanning (DDA) followed by parallel reaction monitoring (PRM), and the efficiency of acquiring MS2 fragment ions of target compounds was significantly improved. Based on the integrated strategy above, 210 lycibarbarspermidine derivatives were detected and identified from Lycii Fructus, and in particular, 170 potential new compounds were structurally characterized. The integrated strategy improved the sensitivity of detection and the coverage of low-response components, and it is expected to be a promising pipeline for discovering new compounds.

Metabolite Extraction from RPE Cells and Retinas Related to Retinitis Pigmentosa.

The application of metabolomics in ophthalmology helps to identify new biomarkers and elucidate disease mechanisms in different eye diseases, as well as aiding in the development of potential treatment options. Extracting metabolites successfully is essential for potential further analysis using mass spectrometry. In this chapter, we describe how to extract metabolites from a variety of sources including (1) cells on a dish, (2) cell culture medium, and (3) tissues in vivo with and without stable isotope tracers. Samples prepared using this protocol are suitable for a range of downstream mass spectrometry analyses and are stable in solvent for weeks at -80 °C.

A quantitative LC-MS method to determine surface contamination of antineoplastic drugs by wipe sampling.

The main objective was to develop a wipe sampling test to measure surface contamination of the most frequently used antineoplastic drugs (ADs) in Swedish healthcare and, furthermore, to develop an analysis method sensitive enough to assess low levels of contamination. Two wipe sampling tests with separate sample processing methods assessing (i) cyclophosphamide (CP), ifosfamide (IF), 5-fluorouracil (5-FU), etoposide (ETO), gemcitabine (GEM) and cytarabine (CYT) (Wipe Test 1); and (ii) GEM, CYT and methotrexate (MTX) (Wipe Test 2), respectively, were developed by optimization of absorption and extraction efficiencies using different wipe tissue materials, tissue wetting solution, and extraction solvents. A fast liquid chromatography tandem mass spectrometry method was developed for simultaneous detection of the studied ADs. The limit of quantification for the method was between 0.04 to 2.4 ng/wipe sample (0.10 to 6.1 pg/cm2 for an area of 400 cm2) and at 50 ng/sample the within-day precision was between 1.3 and 15%, and the accuracy between 102 and 127%. Wipe Test 1 was applied in an assessment of cleaning efficiency of five different cleaning solutions (formic acid, water, sodium hydroxide, ethanol, and sodium dodecyl sulfate (SDS) for removal of ADs from surfaces made of stainless steel or plastic. For CP, IF, 5-FU, GEM, and CYT 92% of the AD were removed regardless of surface and cleaning solution. In conclusion, a user-friendly assessment method to measure low levels of seven ADs in the work environment was developed and validated. Assessment of the decontamination efficiency of cleaning solutions concerning removal of ADs from stainless steel showed that efficiencies differed depending on the AD with water being the least effective cleaning agent. The results suggests that a combination of different cleaning agents including detergent and a solution with an organic component would be optimal to efficiently remove the measured ADs from surfaces in the workplace.

A novel simple and sensitive approach for determination of 1,1-dimethylhydrazine in aqueous samples by high performance liquid chromatography with ultraviolet and tandem mass spectrometric detection after derivatization with unsubstituted aromatic aldehydes.

In this work, simple, rapid and highly sensitive method of hazardous chemical 1,1-dimethylhydrazine (unsymmetrical dimethylhydrazine, UDMH) determination based on pre-column derivatization with unsubstituted aromatic aldehydes and reversed-phase high performance liquid chromatography-ultraviolet-tandem mass spectrometry (RP HPLC-UV-MS/MS) has been developed. Along with benzaldehyde, commercially available aromatic aldehydes, namely: 2-naphthaldehyde, 2-pyridinecarboxaldehyde, and 2-quinolinecarboxaldehyde, were used as derivatizing reagents in the analysis of hydrazines for the first time. The reactions were studied in a wide pH range by varying reaction time and other conditions. A slightly alkaline pH 9 was shown to be optimal for the derivatization of UDMH by aromatic aldehydes. The quantitative yield of derivatization products under the established conditions was confirmed by HPLC analysis with amperometric detection. For all studied reagents, wide linear ranges of concentrations (0.01-1000 µg/L) in natural water samples were observed. The limits of detection for UDMH in natural water were in the 3.7-130 ng/L range. 2-Quinolinecarboxaldehyde was selected as the most appropriate reagent for HPLC-UV-MS/MS determination of UDMH. In case of using this reagent, the accuracy was in the range of 97-102%, and precision, expressed as RSD was less than 8%. The developed approach does not require laborious stages of pre-concentration and isolation of UDMH from natural water components.

Proteomic Analysis of Porcine-Derived Collagen Membrane and Matrix.

Collagen membranes and matrices being widely used in guided bone regeneration and soft tissue augmentation have characteristic properties based on their composition. The respective proteomic signatures have not been identified. Here, we performed a high-resolution shotgun proteomic analysis on two porcine collagen-based biomaterials designed for guided bone regeneration and soft tissue augmentation. Three lots each of a porcine-derived collagen membrane and a matrix derived from peritoneum and/or skin were digested and separated by nano-reverse-phase high-performance liquid chromatography. The peptides were subjected to mass spectrometric detection and analysis. A total of 37 proteins identified by two peptides were present in all collagen membranes and matrices, with 11 and 16 proteins being exclusively present in the membrane and matrix, respectively. The common extracellular matrix proteins include fibrillar collagens (COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL5A3, COL11A2), non-fibrillar collagens (COL4A2, COL6A1, COL6A2, COL6A3, COL7A1, COL16A1, COL22A1), and leucine-rich repeat proteoglycans (DCN, LUM, BGN, PRELP, OGN). The structural proteins vimentin, actin-based microfilaments (ACTB), annexins (ANXA1, ANXA5), tubulins (TUBA1B, TUBB), and histones (H2A, H2B, H4) were also identified. Examples of membrane-only proteins are COL12A1 and COL14A1, and, of matrix only proteins, elastin (ELN). The proteomic signature thus revealed the similarities between but also some individual proteins of collagen membrane and matrix.

Solid phase microextraction of polycyclic aromatic hydrocarbons from water samples by a fiber coated with covalent organic framework modified graphitic carbon nitride.

A covalent organic framework modified graphitic carbon nitride (g-C3N4@TpBD) was synthesized by modifying the graphitic carbon nitride (g-C3N4) with a covalent organic framework (COF-TpBD). The synthesis conditions including the mass ratio between g-C3N4 and benzidine (BD), solvent type, reaction temperature and reaction time were optimized. Under the optimal synthetic conditions, a novel spiny dendritic g-C3N4@TpBD adsorbent was obtained. The g-C3N4@TpBD was then coated on stainless-steel wire by sol-gel technique and the coated fiber was used for the solid phase microextraction of polycyclic aromatic hydrocarbons prior to gas chromatography-mass spectrometric detection. The established method was successfully applied to determine eight PAHs in six environmental water samples. Under the optimal extraction conditions, a wide linear quantification range for the analytes was obtained from 0.07 to 60.0 ng mL-1 with the coefficients of determination varying from 0.9979 to 0.9998, and the limits of detection (S/N = 3) ranged from 0.02 to 0.05 ng mL-1. The relative recoveries of the analytes for the six environmental water samples at the spiked concentrations of 0.2, 0.5, 3.0 and 30.0 ng mL-1 were between 83.6% and 118% with the relative standard deviations ranging from 2.4% to 11.3%.

Enhancement of multianalyte mass spectrometry detection through response surface optimization by least squares and artificial neural network modelling.

In this work, the use of design of experiments and posterior data modelling by artificial neural network (ANN) and least squares (LS) is presented as a suitable analytical tool for the performance optimization of a tandem mass spectrometric detector coupled to ultra-high performance liquid chromatography for the analysis of seventeen veterinary drugs. Firstly, a central composite design was built considering as factors the cone, capillary, extractor and radio frequency voltages of the mass spectrometer in order to obtain a proper combination to improve the sensitivity of the method. Secondly, a one factor design considering the collision voltage was built to define the adequate voltage for each daughter ion. The response surface methodology (RSM) was then applied, and the prediction capability of ANN and LS were compared. As conclusion, the ANN modelling provided better results than LS, both in terms of the ANOVA and predicted areas results. The accuracy of the model prediction was between 85 and 125%, confirming that the estimates of the model were correct, and endorsing the optimization procedure as a suitable way to gather excellent results. The suitability of the new approach and its implications on the simultaneous analysis of seventeen veterinary drugs by ultra-high liquid chromatography coupled to tandem mass spectrometry detection are discussed.

Portable and low-cost biosensor towards on-site detection of diclofenac in wastewater.

Wastewater treatment plants are the main release sources of pharmaceutical compounds present in surface waters. Even at low concentrations, many of these substances have long-term adverse effects on the environment. For an efficient control of pharmaceutical removal, a real-time recognition is a prerequisite. Currently, quantification of such compounds is done in special equipped laboratories and is rather time-consuming and expensive. Here, we introduce a novel biosensor for the detection of the pharmaceutical compound diclofenac, which can be produced with low costs, is easy in handling and can be applied directly on-site. Recognition of diclofenac is based on genetically engineered yeast cells which produce green fluorescent protein in a diclofenac concentration-dependent manner. Centerpiece of the sensor is a foil-based microfluidic flow cell, which allows supply with nutrient solution and analyte while preventing loss of reporter cells. Readout of data is accomplished by a newly developed spectrometric detection unit. With this device, we are able to determine diclofenac concentrations in a range from 10 to 50 µM.

Gas chromatography with tandem cold electron ionization mass spectrometric detection and vacuum ultraviolet detection for the comprehensive analysis of fentanyl analogues.

Fentanyl and its derivatives are amongst the ever-growing list of emerging drugs which are impinging on current traditional analytical techniques employed in forensic laboratories. To avoid current regulations, fentanyl analogues are being illicitly synthesized by slight alterations of functional groups to the fentanyl skeleton leading to inaccurate identifications, thus posing the greatest challenge to laboratories. In this study, a novel analytical technique is presented in which gas chromatography (GC) is interfaced with both cold electron ionization mass spectrometric (cold EI MS) and vacuum ultraviolet (VUV) detection by the means of a flow splitter for the simultaneous qualitative and quantitative analysis of twenty-four fentanyl analogues, including seven sets of positional isomers. For most of the twenty-four analogues, enhanced molecular ions were obtained with at least 1% intensity relative to base peak. In addition to enhanced molecular ions, the GC-cold EI MS maintained fragmentation pathways observed by GCMS with classical electron ionization. For the most part, VUV detection resulted in unique VUV spectra for fentanyl analogues including positional isomers. The combination of these two complementary detectors in tandem with the high resolving power of the gas chromatograph, allows for higher confidence in analyte identification by the combination of retention times, cold EI mass spectra and VUV spectra. The preferred method for quantitation was based on VUV detection and offered excellent determination coefficients (R2≥0.999) for most analytes over two orders of magnitude dynamic range, without the need for deuterated internal standards. For both run-to-run and day-to-day repeatability studies, at moderate solute concentrations, the correct fentanyl related compound was identified in almost every instance from a library containing all the fentanyl analogues plus hundreds of other analytes.

Effect of Low Temperature Cultivation on the Phytochemical Profile and Bioactivity of Arctic Plants: A Case of Dracocephalum palmatum.

The influence of climatic factors, e.g., low temperature, on the phytochemical composition and bioactivity of the arctic plant Dracocephalum palmatum Steph. ax Willd. (palmate dragonhead), a traditional food and medical herb of Northern Siberia, was investigated. D. palmatum seedlings were grown in a greenhouse experiment at normal (20 °C, NT) and low (1 °C, LT) temperature levels and five groups of components that were lipophilic and hydrophilic in nature were characterized. The analyses indicated that D. palmatum under NT demonstrates high content of photosynthetic pigments, specific fatty acid (FA) profile with domination of saturated FA (53.3%) and the essential oil with trans-pinocamphone as a main component (37.9%). Phenolic compounds were identified using a combination of high performance liquid chromatography with diode array detection and electrospray ionization mass-spectrometric detection (HPLC-DAD-ESI-MS) techniques, as well as free carbohydrates and water soluble polysaccharides. For the first time, it was established that the cold acclimation of D. palmatum seedlings resulted in various changes in physiological and biochemical parameters such as membrane permeability, photosynthetic potential, membrane fluidity, leaf surface secretory function, reactive oxygen species-antioxidant balance, osmoregulator content and cell wall polymers. In brief, results showed that the adaptive strategy of D. palmatum under LT was realized on the accumulation of membrane or surface components with more fluid properties (unsaturated FA and essential oils), antioxidants (phenolic compounds and enzymes), osmoprotectants (free sugars) and cell wall components (polysaccharides). In addition, the occurrence of unusual flavonoids including two new isomeric malonyl esters of eriodictyol-7-O-glucoside was found in LT samples. Data thus obtained allow improving our understanding of ecophysiological mechanisms of cold adaptation of arctic plants.

Mass spectrometric detection combined with bioinformatic analysis identified possible protein markers and key pathways associated with bladder cancer.

We aimed to find possible protein markers and key pathways related to bladder cancer. In total, we extracted three bladder cancer tissues and three paracancerous tissues from Jiangsu Provincial People's Hospital Urology Department, and performed mass spectrometric detection with Q Exactive. Subsequently, we screened the differentially expressed proteins in the disease group and the normal group using the LIMMA package, and performed functional enrichment analyses using DAVID. Further, we constructed protein-protein interaction (PPI) networks with Cytoscape software, and analyzed modules with ClusterONE. In total, 165 differentially expressed proteins including 19 upregulated and 146 downregulated ones were obtained. ACTA2 (Actin, Alpha 2, Smooth Muscle, Aorta), ACTN1 (Actinin, Alpha 1), and VCL (Vinculin) were significant nodes with higher degrees in the PPI network. These three nodes were also hub nodes in module 2. Besides, functional enrichment analysis suggested that ECM-receptor interaction and focal adhesion were significant pathways, and these two pathways were also enriched in three network modules. In addition, ACTN1 and VCL were enriched in the focal adhesion pathway in module 2. Thus, ACTA2, ACTN1, and VCL may play important roles in bladder cancer progression and may be protein markers for this disease. The ECM-receptor interaction pathway and the focal adhesion pathway may be involved in the progression of bladder cancer. Furthermore, ACTN1 and VCL may play roles in bladder cancer development, partly via the focal adhesion pathway.

An improved method for the analysis of 2-aminoacetophenone in wine based on headspace solid-phase microextraction and heart-cut multidimensional gas chromatography with selective detection by tandem mass spectrometry.

Heart-cut multidimensional gas chromatography coupled to selective mass spectrometric detection (H/C MDGC-MS-MS) was shown to be a suitable combination for analysis of the key component for the wine off-flavor named "atypical aging off-flavor" (german: "Untypische Alterungsnote", UTA), 2-aminoacetophenone (2-AAP). Headspace solid phase microextraction (HS-SPME) was applied as an easy to automate sample preparation step suiting routine control situations. During method development two critical aspects were found, of which one is the chemical degradation of the neat substance, leading to signal reduction and a false response behavior. A second aspect is the pH stability of the deuterated isotopologue 1-(2-aminophenyl)-2,2,2-trideuterio-ethanone (2-AAP-d3) used for quantification via a stable isotope dilution assay. Despite an earlier suggestion to increase the extractable amount of 2-AAP and its isotopic standard in the headspace, alkalization of 2-AAP-d3 containing aqueous samples were found to be critical. In such a milieu a deuterium exchange can then cause erroneous quantitative results. The method proposed in our study thus uses native wine pH conditions and proved to be suitable for routine control with respect to the detection of the "atypical aging off-flavor" in wine at concentration levels below its sensory threshold of about 1µgL-1. Good linearity was obtained in the calibrated range from 0.1-8µgL-1 (y=1,2338x+0,1029, R=0,9985) with limits of detection or quantification being 0.01 and 0.14µg/L, respectively.

Nonaqueous Capillary Electrophoresis Mass Spectrometry.

The term nonaqueous capillary electrophoresis (NACE) commonly refers to capillary electrophoresis with purely nonaqueous background electrolytes (BGE). Main advantages of NACE are the possibility to analyze substances with very low solubility in aqueous media as well as separation selectivity that can be quite different in organic solvents (compared to water)-a property that can be employed for manipulation of separation selectivities. Mass spectrometry (MS) has become more and more popular as a detector in CE a fact that applies also for NACE. In the present chapter, the development of NACE-MS since 2004 is reviewed. Relevant parameters like composition of BGE and its influence on separation and detection in NACE as well as sheath liquid for NACE-MS are discussed. Finally, an overview of the papers published in the field of NACE-MS between 2004 and 2014 is given. Applications are grouped according to the field (analysis of natural products, biomedical analysis, food analysis, analysis of industrial products, and fundamental investigations).

Enantiodifferentiation of 1,2-propanediol in various wines as phenylboronate ester with multidimensional gas chromatography-mass spectrometry.

Native concentrations and enantiomeric distribution of 1,2-propanediol in various wines were studied in order to evaluate its merits as a potential marker for aroma adulteration in wine. Heart-cut multidimensional gas chromatography coupled to mass spectrometry was applied to analyze 1,2-propanediol after salting-out of the polar phase, derivatization with phenyl boronic acid, and extraction with cyclohexane. The enantiomeric separation of the derivative was achieved with heptakis-(6-O-tert. butyl dimethylsilyl-2,3-di-O-acetyl)-ß-cyclodextrin as the chiral selector. In all authentic wines studied, 1,2-propanediol showed a high enantiomeric ratio in favor of the (R)-enantiomer, proving its potential as a marker for the adulteration with flavor extracts based on industrial 1,2-propandiol as solvent. Usually, concentrations varied between 15 and 100 mg/L. Higher values (up to 170 mg/L) were found in wines made with high amounts of dry berries. However, despite the higher concentrations of 1,2-propanediol in such wines, no apparent influence on the enantiomeric distribution could be detected. Graphical Abstract Detection of fraudulent aromatization of wines by enantiodifferentiation of 1,2-propanediol as its phenylboronate ester.

Capillary isotachophoresis with ESI-MS detection: Methodology for highly sensitive analysis of ibuprofen and diclofenac in waters.

The possibilities of reaching higher sensitivity in capillary electrophoretic analyses of complex samples with ESI-MS detection were investigated on the example of analysis of diclofenac and ibuprofen in waters. The applied separation approach is based on application of isotachophoresis that ensures permanent stacking of analytes until they reach the detector. Investigation of the possibilities of MS detector optimization have shown that optimization of fragmentor voltage and working in the SIM mode with collection of data for multiple fragments both increases the method specificity and approx. doubles its sensitivity. Combination with an offline SPE preconcentration step resulted in very high sensitivity of the described methodology with a reached LOD below 2 × 10(-12) M, corresponding to analyte levels of 0.6 ng L(-1) of diclofenac and 0.4 ng L(-1) of ibuprofen. The results demonstrate that CE-MS, particularly when performed in the ITP mode, has the potential to reach sensitivities comparable to HPLC-MS.

Rapid and sensitive methodology for determination of ethyl carbamate in fortified wines using microextraction by packed sorbent and gas chromatography with mass spectrometric detection.

This work presents a new methodology to quantify ethyl carbamate (EC) in fortified wines. The presented approach combines the microextraction by packed sorbent (MEPS), using a hand-held automated analytical syringe, with one-dimensional gas chromatography coupled with mass spectrometry detection (GC-MS). The performance of different MEPS sorbent materials was tested, namely SIL, C2, C8, C18, and M1. Also, several extraction solvents and the matrix effect were evaluated. Experimental data showed that C8 and dichloromethane were the best sorbent/solvent pair to extract EC. Concerning solvent and sample volumes optimization used in MEPS extraction an experimental design (DoE) was carried out. The best extraction yield was achieved passing 300 µL of sample and 100 µL of dichloromethane. The method validation was performed using a matrix-matched calibration using both sweet and dry fortified wines, to minimize the matrix effect. The proposed methodology presented good linearity (R(2)=0.9999) and high sensitivity, with quite low limits of detection (LOD) and quantification (LOQ), 1.5 µg L(-1) and 4.5 µg L(-1), respectively. The recoveries varied between 97% and 106%, while the method precision (repeatability and reproducibility) was lower than 7%. The applicability of the methodology was confirmed through the analysis of 16 fortified wines, with values ranging between 7.3 and 206 µg L(-1). All chromatograms showed good peak resolution, confirming its selectivity. The developed MEPS/GC-MS methodology arises as an important tool to quantify EC in fortified wines, combining efficiency and effectiveness, with simpler, faster and affordable analytical procedures that provide great sensitivity without using sophisticated and expensive equipment.

On the use of quadrupole mass spectrometric detection for flow modulated comprehensive two-dimensional gas chromatography.

Flow modulated comprehensive two-dimensional gas chromatography with simultaneous flame ionization detection (FM GC×GC-FID) and quadrupole mass spectrometric detection (FM GC×GC-qMSD) was applied to the analysis of a quantitative reference standard sample covering the C5-C14 hydrocarbon range and a kerosene sample. This study reports the influence of the data acquisition frequency (DAF) of the qMSD on the qualitative and quantitative data. The DAF was varied between 5.27 and 25.45Hz. In GC×GC-qMSD, the characterization of peaks is getting worse with decreasing qMSD frequencies which deteriorate the quality of the 2D contour plots of the separations. It is, however, demonstrated that the improved characterization of the peaks at high qMSD frequencies is combined with loss in sensitivity and in spectral quality. Dependences of a (2)D hydrocarbon peak area (∑Ai of all corresponding (2)D peaks) on the DAFs show negative slopes which testify the conclusion that the sensitivity of GC×GC-qMSD determinations decreases with the increase of DAF. Consequently, the limits of detection (LOD) and quantification (LOQ) increase with the increase of DAF. Moreover, quality criteria on recorded spectral data also indicate that the lower the DAF is, the higher is the spectral quality.

Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, ß-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 µM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.