Persistence, dietary and ecological risk assessment of indoxacarb residue in/on tomato and soil using GC-MS.

An efficient single quadrupole gas chromatography with mass spectrometry method was developed and validated for the determination of indoxacarb residues in tomato and soil. Residues were extracted from the samples using acetonitrile as extracting solvent and the extracts were purified through primary secondary amine and graphitized carbon black. Recoveries were obtained in the range of 92.12-110.51% with the relative standard deviation of 1.32-4.32%. Indoxacarb dissipated with half-life of 3.12-3.21 and 1.24-1.35d for tomato and soil, respectively following doses of indoxacarb 14.5% SC at 60, 90 and 120 g.a.i./ha. Safe waiting periods were found to be 1-3d. The residues were removed from tomato fruit was in the range of 16.73 to 54.32% using simple decontamination approaches. The present study suggest that the use of indoxacarb in tomato at recommended dose, does not seem to pose any dietary risk to the consumers. The soil RQ values indicated low level of risk to earthworms and arthropods.

Quality by Design oriented development of hydrophilic interaction liquid chromatography method for the analysis of amitriptyline and its impurities.

This paper presents integration of Quality by Design concept in the development of hydrophilic interactions liquid chromatographic methods for analysis of amitriptyline and its impurities (A, B, C, and F). This is the first time that HILIC method for amitriptyline and its impurities is developed. Using QbD concept, it is possible to design a robust method and incorporate quality directly into its development. QbD concept in combination of Design of Experiments methodology (DoE) enables creation of well-defined design space. In this study, for method optimization a Box-Behnken design was used to test the effect of acetonitrile content, buffer concentration and pH of water phase on critical system responses such as retention factor of impurity A, resolution between impurity B and impurity C, amitriptyline peak asymmetry factor and retention time of last eluted impurity F. The defined mathematical models and Monte Carlo simulations were used to identify the design space. For robustness testing, fractional factorial design was applied. Optimal chromatographic conditions were the analytical column ZORBAX NH2 (250 mm x 4.6 mm, 5 µm particle size); mobile phase consisted of acetonitrile-water phase (60 mM ammonium acetate, pH adjusted to 4.5 with glacial acetic acid) (92.5:7.5 v/v); column temperature 30 °C, mobile phase flow rate 1 mL min-1, wavelength of detection 254 nm. Finally, method was fully validated and applicability of the method in tablet analysis was confirmed.

Analysis of 4(5)-methylimidazole in soy sauce by a quick, easy, cheap, effective, rugged, and safe approach and liquid chromatography-mass spectrometry.

4(5)-Methylimidazole (4(5)-MI) is a potential carcinogen with low molecule weight, highly polarity, and weak basicity. The traditional way to extract and clean-up 4(5)-MI in soy sauce using solid phase extraction is tedious and time consuming. Here we proposed a method for the determination of 4(5)-MI in soy sauce by combining a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction with liquid chromatography-mass spectrometry analysis. The impacts of solution pH, water addition, and cleanup procedure on 4(5)-MI extraction efficiency were studied. An optimized sample preparation approach involved a single step liquid-liquid extraction between acetonitrile and soy sauce under alkaline conditions, followed by primary and secondary amine clean-up. The analytical method was validated with soy sauce at three spiking levels (10, 50, 500 ng/g). The method recovery (96.2-107%) and intra-day/inter-day precision (4.1-8.4%/6.9-11.7%) were satisfactory. The method quantification limit was 10 ng/g. The developed method was successfully applied for the determination of 4(5)-MI in fourteen commercial soy sauces from local markets. The results obtained in this work suggests that the method is suitable for the analysis of 4(5)-MI at low concentrations in high-salting and protein-containing soy sauce matrix.

Matrix solid-phase dispersion associated to gas chromatography for the assessment in honey bee of a group of pesticides of concern in the apicultural field.

A method based on matrix solid-phase dispersion (MSPD) associated to gas chromatography-flame photometric detection (GC-FPD), GC-electron capture detection (GC-ECD) and GC-mass spectrometry (GC-MS) for confirmation purposes, was developed for the determination of a representative group of twelve pesticides in honeybee with particular concern in the apicultural field (fipronil, thiamethoxam, acetamiprid, acrinathrin, metamidophos, dimetoathe, diazinon, chlorpyrifos, methidathion, profenophos, azinphos methyl and coumaphos). Factors influencing the extraction efficiency of MSPD were investigated and optimized through response surface method. The use of octadecylsilyl (C18) sorbent combined with a florisil clean-up and acetonitrile-methanol (99:1) elution was the optimal condition for the extraction of the selected pesticides. Under this condition the recovery of pesticides at the limit of quantification of the method (0.007 to 0.050 µg g-1) ranged from 68 to 102% with RSDs for within-laboratory reproducibility ≤20%. The proposed method was applied to the analysis of honeybees collected in 68 field hives from areas of great apicultural and agricultural development in central Chile. In 65% of these samples eight different pesticides were detected. Pesticides most frequently found were chlorpyrifos (34% of the samples, <0.017-0.067 µg g-1), acrinathrin (32% of the samples, <0.020-0.026 µg g-1) and diazinon (10% of the samples at values <0.015 µg g-1). The incidence of these pesticides in bees can be related to their high employ in central Chile, use to combat the varroosis in hives and hydrophobicity.

A modified quick, easy, cheap, effective, rugged, and safe cleanup method followed by liquid chromatography-tandem mass spectrometry for the rapid analysis of perchlorate, bromate and hypophosphite in flour.

A selective, sensitive and useful method, based on modified QuEChERS cleanup combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the negative-ion electrospray ionization (ESI-) mode, was developed and validated for the simultaneous determination of three inorganic anions including perchlorate (ClO4-), bromate (BrO3-) and hypophosphite (H2PO2-) in flour. The extraction parameters and LC-MS/MS conditions were optimized by single-factor experiment and sorbent combination in modified QuEChERS clean-up was optimized through response surface analysis. Three target analytes were separated on a normal-phase Phenomenex Luna Silica (2) column (150mm×2.0mm, 5µm, 100Å) with the mobile phase of a mixture of 5mmol/L ammonium acetate water solution and acetonitrile, detected by MS/MS under multiple reaction monitoring and quantified by external standard method. The developed method was validated in terms of the sensitivity, linearity, accuracy and precision, and matrix effect. The method showed a good linearity (R2>0.999) for all analytes in their respective concentration ranges. The ILOQs and MLOQs for perchlorate, bromate and hypophosphite were 0.1, 0.5, 5.0µg/L and 2.0, 6.0, 60.0µg/L, respectively. The average recoveries of three target analytes from the negative samples spiked at three different concentrations were in a range from 84.6% to 104.9%. The intra-day precision (n=6) and inter-day precision (n=5) of the target analytes were in the ranges of 2.9%-6.9% and 6.4%-8.2%. The matrix effect of this method was observed between 0.83 and 1.17 and was acceptable. The validated method was successfully applied to determine the concentrations of these inorganic anions in flour. Results found that perchlorate and hypophosphite were detected in 33 out of 50 and 7 out of 50 flour samples.

Effective purification of recombinant peptide ligands for GPCR research.

Peptide purification from natural sources and chemical synthesis is cumbersome with various shortcomings such as low yield, high cost of production, error prone, and restricted by nature of amino acids. Though recombinant DNA technology had overcome all these setbacks for larger proteins, it is still a challenge to produce peptides that are salt free and without impurities. Our approach discussed in this chapter deals with easy and effective purification of peptides of varying sizes (up to 10kDa), expressed as fusion proteins in bacterial system. This includes cleavage of fusion affinity tag by "PreScission protease" in volatile buffer followed by selective acetonitrile precipitation of high-molecular-weight tag in order to purify peptides in solution. This method can be used to purify peptides in large scale for various biochemical and physiological studies.

Determination of eight pesticides in Lycium barbarum by LC-MS/MS and dietary risk assessment.

A LC-MS/MS method for determination of eight pesticides (triadimefon, sulfoxaflor, flusilazole, tebuconazole, difenoconazole, amitraz, azoxystrobin, and thiophanate-methyl) in Lycium barbarum was established. The samples were extracted with acetonitrile, and then cleaned up by primary secondary amine. The extracts were diluted with 0.1% formic acid in water. The results showed that at the fortified levels of 0.01-10mg/kg, the average recoveries of these pesticides ranged from 82.1% to 96.2% with the relative standard deviations lower than 7%. The half-lives of eight pesticides were 1.3-5.0days in Lycium barbarum fruits. The pre-harvest interval of all pesticides mentioned above were investigated. Tebuconazole (14days), sulfoxaflor (14days) and flusilazole (28days) have longer pre-harvest interval than the others which have 7days. The dietary risks, assessed as hazard quotients, were far below 100%. The results showed that the eight pesticides applied to Lycium barbarum were comparably safe for the consumer.

Quantitative assessment of methyl-esterification and other side reactions in a standard propionylation protocol for detection of histone modifications.

Histone modifications play an important role in regulating chromatin stability and gene expression, but to date, investigating them remains challenging. In order to obtain peptides suitable for MS-based analysis, chemical derivatization of N-terminus and lysine residues by propionic anhydride is commonly performed. Several side reactions (methyl-esterification, amidation, solvolysis, overpropionylation, and missed propionylation) during propionylation protocols have been described, yet their relative abundances remain vague. Because methyl-esterification could interfere with correct interpretation of the modification pattern, it is essential to take measures to avoid it. Here we present in-depth quantitative analyses of methyl-esterification and the other side reactions in a standard propionylation protocol containing methanol, and when replacing methanol with isopropanol or acetonitrile. We show that the use of alternative solvents can eliminate methyl-esterification and that even though other side reactions are not prevented, their contribution can be kept relatively small. We also show that replacing methanol can be of importance also in other proteomics methods, such as mixed cation exchange, using methanol under acidic conditions.

A LC-MS/MS method for the determination of stachyose in rat plasma and its application to a pharmacokinetic study.

A sensitive, simple and rapid analytical method based on a liquid chromatography-tandem mass spetrometry (LC-MS/MS) has been established and validated for the determination of stachyose in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Separation of stachyose and nystose (internal standard, IS) was achieved using acetonitrile-water (55:45, v/v) as the mobile phase at a flow rate of 1 ml/min for 6 min on an Asahipak NH2P-50 4E column with an Asahipak NH2P-50G 4A guard column. Detection and quantification were conducted by LC-MS/MS method in the negative ion mode using multiple reaction monitoring (MRM) transitions at m/z [M-H](-) 665.4→383.1 for stachyose and 665.5→485.0 for IS, respectively. The method was linear over the concentration ranges of 100-30000 ng/ml with a lower limit of quantification (LLOQ) of 100 ng/ml. The intra- and inter- day precision were all within 8.7% and the accuracy ranged from 97.2-108.4% and 98.3-102.4%, respectively. Stability studies indicated that stachyose was stable under short-term, long-term and three freeze-thaw storage conditions. The method was successfully applied to a pharmacokinetic study involving pulmonary administration of micronized Rehmannia glutinosa oligosaccharides (RGOS) to rats.

Assessment of lifetime resolution limits in time-resolved photoacoustic calorimetry vs. transducer frequencies: setting the stage for picosecond resolution.

Time-resolved photoacoustic calorimetry (PAC) gives access to lifetimes and energy fractions of reaction intermediates by deconvolution of the photoacoustic wave of a sample (E-wave) with that of the instrumental response (T-wave). The ability to discriminate between short lifetimes increases with transducer frequencies employed to detect the PAC waves. We investigate the lifetime resolution limits of PAC as a function of the transducer frequencies using the instrumental response obtained with the photoacoustic reference 2-hydroxybenzophenone in toluene or acetonitrile. The instrumental response was obtained for a set of transducers with central frequencies ranging from 0.5 MHz up to 225 MHz. The simulated dependence of the lifetime resolution with the transducer frequencies was anchored on experimental data obtained for the singlet state of naphthalene with a 2.25 MHz transducer. The shortest lifetime resolved with the 2.25 MHz transducer was 19 ns and our modelling of the transducer responses indicates that sub-nanosecond lifetimes of photoacoustic transients can be resolved with transducers of central frequencies above 100 MHz.

Straightforward synthesis of radioiodinated Cc-substituted o-carboranes: towards a versatile platform to enable the in vivo assessment of BNCT drug candidates.

Due to their high boron content and rich chemistry, dicarba-closo-dodecaboranes (carboranes) are promising building blocks for the development of drug candidates with application in Boron Neutron Capture Therapy. However, the non-invasive determination of their pharmacokinetic properties to predict therapeutic efficacy is still a challenge. Herein, we have reported the unprecedented preparation of mono-[(125)I] iodinated decaborane via a catalyst-assisted isotopic exchange. Subsequent reactions of the radiolabelled species with acetylenes in acetonitrile under microwave heating yield the corresponding (125)I-labelled, Cc-substituted o-carboranes with good overall radiochemical yields in short reaction times. The same synthetic strategy was successfully applied to the preparation of (131)I-labelled analogues, and further extension to other radioisotopes of iodine such as (124)I (positron emitter) or (123)I (gamma emitter) can be envisaged. Hence, the general strategy reported here is suitable for the preparation of a wide range of radiolabelled Cc-substituted o-carborane derivatives. The labelled compounds might be subsequently investigated in vivo by using nuclear imaging techniques such as Single Photon Emission Computerized Tomography or Positron Emission Tomography.

Influence of activated carbon porosity and surface oxygen functionalities' presence on adsorption of acetonitrile as a simple polar volatile organic compound.

Based on series of porous carbon models, systematic Monte Carlo studies on the adsorption of acetonitrile (as a simple representative of polar volatile organic compounds) were performed. The influence of porosity and chemical composition of the carbon surface on CH3CN adsorption was studied and it was shown that both the factors influenced the adsorption mechanism. A decrease in the pore size and the introduction of oxygen surface groups led to a rise in adsorption energy and to an increase in the filling of accessible volume in the low-pressure part of the isotherm. However, from a practical point of view, it is easier to increase the adsorption by introducing polar groups on the carbon surface than by modifying the porosity.

Development and Validation of a Stability-Indicating Liquid Chromatographic Method for Estimating Olmesartan Medoxomil Using Quality by Design.

The current studies entail systematic quality by design (QbD)-based development of a simple, rapid, sensitive and cost-effective stability-indicating method for the estimation of olmesartan medoxomil. Quality target method profile was defined and critical analytical attributes (CAAs) for the reverse-phase liquid chromatography method earmarked. Chromatographic separation accomplished on a C18 column using acetonitrile and water (containing 0.1% orthophosphoric acid, pH 3.5) in 40 : 60 (v/v) as mobile phase at a flow rate of 1.0 mL/min with UV detection at 243 nm. Risk assessment studies and screening studies facilitated comprehensive understanding of the factors affecting CAAs. The mobile phase ratio and flow rate were identified as critical method parameters (CMPs) and were systematically optimized using face-centered cubic design, evaluating for CAAs, namely peak area, retention time, theoretical plates and peak tailing. Statistical modelization was accomplished followed by response surface analysis for comprehending plausible interaction(s) among CMPs. Search for optimum solution was conducted through numerical and graphical optimization for demarcating the design space. Analytical method validation and subsequent forced degradation studies corroborated the method to be highly efficient for routine analysis of drug and its degradation products. The studies successfully demonstrate the utility of QbD approach for developing the highly sensitive liquid chromatographic method with enhanced method performance.

The activity-integrated method for quality assessment of reduning injection by on-line DPPH-CE-DAD.

A sensitive on-line DPPH-CE-DAD method was developed and validated for both screening and determining the concentration of seven antioxidants of Reduning injection. The pH and concentrations of buffer solution, SDS, ß-CD and organic modifier were studied for the detection of DPPH and seven antioxidants. By on-line mixing DPPH and sample solution, a DPPH-CE method for testing the antioxidant activity of the complex matrix was successfully established and used to screen the antioxidant components of Reduning injection. Then, antioxidant components including caffeic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid were quantified by the newly established CE-DAD method. Finally, the total antioxidant activity and the multiple active components were selected as markers to evaluate the quality of Reduning injection. The results demonstrated that the on-line DPPH-CE-DAD method was reagent-saving, rapid and feasible for on-line simultaneous determination of total pharmacological activity and contents of multi-components samples. It was also a powerful method for evaluating the quality control and mechanism of action of TCM injection.

Development, optimization and validation of a multimethod for the determination of 36 mycotoxins in wines by liquid chromatography-tandem mass spectrometry.

A fast and efficient multimethod for the determination of 36 mycotoxins in wine, using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), was developed, optimized, validated and implemented in routine analysis. A simplified, quick extraction was performed with acetonitrile, derived from the QuEChERS (quick, easy, cheap, effective, rugged and safe) approach, which was traditionally developed for pesticides analysis. This study aimed at a single extraction and chromatographic separation for 36 mycotoxins. Optimization tests were performed to find the proper ratio of wine: water and extraction solvent and the need for an additional buffering step with ammonium formate/formic acid and a dispersive SPE cleanup with various sorbents. The dSPE steps did not show significant improvement in analysis results, therefore, it was not applied in the final method to be validated. The mycotoxins were separated and detected on a UPLC-MS/MS system, used in the ESI positive ionization mode. The various mycotoxins were divided in three different concentration level groups, according to their sensitivity in UPLC-MS/MS. The validation was performed by analyzing recovery samples at three different spike levels with six replicates (n=6) at each level. Linearity (r(2)) of calibration curves, accuracy (recovery %), instrument limits of detection and method limits of quantification (LOD and LOQ), precision (RSD%) and matrix effects (%) were determined for each individual mycotoxin. From the 36 mycotoxins analyzed by UPLC-MS/MS (ESI+), 35 showed average recoveries in the range 70-120%, and 86% of these with a RSD≤20% at the lowest spike level (for Group I, II and III, respectively, 1, 50 and 10 µg kg(-1)). The higher spike levels showed even better results. Only nivalenol could not be quantified at any concentration level. The method LOQ for 86% of the mycotoxins studied was the lowest spike level tested. The matrix effect observed was low for most mycotoxins analyzed and had no significant influence on the analytical results obtained. The developed procedure was applied successfully in routine analysis in a survey of wine samples originating from different countries.

Assessment of the complementarity of temperature and flow-rate for response normalisation of aerosol-based detectors.

The solvent dependency of the detection response is a major limitation of corona-charged aerosol detection (C-CAD). The present study empirically investigates the utility of temperature and flow-rate gradients to overcome solvent gradient limitations of C-CAD. In preliminary flow-injection investigations, it is demonstrated that the response of C-CAD remains relatively unaltered with variations in flow-rate when used with water-rich eluents. Based on these findings two separation approaches were developed and their utility for C-CAD response normalisation was demonstrated using a mixture of eight analytes. In the first approach the use of a solvent gradient is replaced with a temperature gradient performed under isocratic mobile phase conditions. Detection response is further enhanced by mixing a secondary stream of pure acetonitrile with the column effluent, yielding a 3-fold increase in detection response. In the second approach, flow-rate programming is used to improve speed of isocratic-temperature gradient separation. The use of simultaneous variation in flow-rate and column temperature reduced the separation time by 30%, with relatively uniform analyte response. Lastly, an inverse-gradient solvent compensation approach was used to evaluate the response homogeneity and the applicability of the above approaches for quantitative analysis. Good peak area reproducibility (RSD%<15%) and linearity (R(2)>0.994, on a log-scale) over the sample mass range of 0.1-10 µg was achieved. The response deviation across the mixture of eight compounds at seven concentration levels was 6-13% compared to 21-39% when a conventional solvent gradient was applied and this response deviation was comparable to that obtained in the inverse gradient solvent compensation approach. Finally, applicability of these approaches for typical pharmaceutical impurity profiling was demonstrated at a concentration of 5 µg/mL (0.1% of the principal compound).

High-throughput determination of nonsteroidal anti-inflammatory drugs in human plasma by HILIC-MS/MS.

A simple and sensitive method was developed and validated here for the analysis of thirteen nonsteroidal anti-inflammatory drugs (NSAIDs) in human plasma samples by hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry (MS/MS). A small volume of plasma (20µL) spiked with compounds was diluted with 80µL of 10-mM ammonium acetate followed by a simple protein precipitation with 400µL of acetonitrile. After centrifugation, the clear supernatant extract was directly injected into the HILIC-MS/MS, without any solvent evaporation and reconstitution steps. The chromatographic separation of the NSAIDs was achieved on a Unison UK-Amino HILIC column (50mm×3mm i.d., particle size 3µm) with a linear gradient elution system composed of 10mM ammonium acetate (pH 6.8) and acetonitrile at a flow rate of 0.4mL/min. The mass spectra obtained by HILIC-MS showed base peak ions due to [M+H](+) for indomethacin, oxaprozin, ketoprofen, alminoprofen, zaltoprofen, tiaprofenic acid, pranoprofen, and ketoprofen-d3 and due to [M-H](-) for etodolac, ibuprofen, diclofenac, fenoprofen, loxoprofen, naproxen, and ibuprofen-d3. Recoveries of these thirteen NSAIDs in plasma were 34.8-113% and the lower limits of quantitation were 0.125-1.25µg/mL. The intra- and interday coefficient of variations for all drugs in plasma were less than 14.6%. The data obtained from actual plasma determinations of zaltoprofen, ibuprofen, and diclofenac are also presented.

A cost-effective and practical polybenzanthrone-based fluorescent sensor for efficient determination of palladium (II) ion and its application in agricultural crops and environment.

A highly selective and sensitive fluorescent chemosensor suitable for practical measurement of palladium ion (Pd(2+)) in agricultural crops and environment samples has been successfully fabricated using polybenzanthrone (PBA). PBA was facilely electrosynthesized in the mixed electrolyte of acetonitrile and boron trifluoride diethyl etherate. The fluorescence intensity of PBA showed a linear response to Pd(2+) in the concentration range of 5 nM-0.12 mM with a detection limit of 0.277 nM and quantification limit of 0.925 nM. Different compounds existing in agricultural crops and environment such as common metal ions, anions, natural amino acids, carbohydrates, and organic acids were used to examine the selectivity of the as-fabricated sensor, and no obvious fluorescence change could be observed in these interferents and their mixtures. A possible mechanism was proposed that the coordination of PBA and Pd(2+) enhance the aggregation of polymer chains, which led to a significant quenching of PBA emission, and this was further confirmed by absorption spectra monitoring and transmission electron microscopy. The excellent performance of the proposed sensor and satisfactory results of the Pd(2+) determination in practical samples suggested that the PBA-based fluorescent sensor for the determination of Pd(2+) will be a good candidate for application in agriculture and environment.

Mobile phase pH and organic modifier in reversed-phase LC-ESI-MS bioanalytical methods: assessment of sensitivity, chromatography and correlation of retention time with in silico logD predictions.

BACKGROUND: The aim of the work described herein was to undertake a systematic investigation of the effect of mobile phase pH and organic modifier in typical reversed-phase LC-MS methods with regard to ESI-MS response, chromatographic performance and correlation of retention time with in silico logD predictions. RESULTS: For the test set of pharmaceutical analytes investigated, ESI-MS response was generally greater when employing methanol rather than acetonitrile as the organic modifier, and increases of up to tenfold were observed dependent on the pH-buffered mobile phase employed. Deleterious effects on chromatographic performance of protonated basic analyte were observed under conditions of neutral to weakly basic pH. A qualitative correlation between plots of predicted logD and observed retention time against pH was demonstrated. CONCLUSION: In the absence of a simple and/or predictive dependence of analyte ESI-MS response on the mobile phase pH, a practical evaluation should be undertaken when absolute sensitivity is paramount. The use of in silico predictions of analyte logD to direct the development of bioanalytical assays is broadly valid, but further scrutiny is recommended in predicting the retention of ionized analyte.