Validation of a High-Performance Liquid Chromatography with Photodiode Array Detection Method for the Separation and Quantification of Antioxidant and Skin Anti-Aging Flavonoids from Nelumbo nucifera Gaertn. Stamen Extract.

Nelumbo nucifera Gaertn., or the so-called sacred lotus, is a useful aquatic plant in the Nelumbonaceae family that has long been used to prepare teas, traditional medicines as well as foods. Many studies reported on the phytochemicals and biological activities of its leaves and seeds. However, to date, only few studies were conducted on its stamen, which is the most important ingredient for herbal medicines, teas and other phytopharmaceutical products. Thus, this present study focuses on the following: (1) the application of high-performance liquid chromatography with photodiode array detection for a validated separation and quantification of flavonoids from stamen; (2) the Nelumbo nucifera stamen's in vitro and in cellulo antioxidant activities; as well as (3) its potential regarding the inhibition of skin aging enzymes for cosmetic applications. The optimal separation of the main flavonoids from the stamen ethanolic extract was effectively achieved using a core-shell column. The results indicated that stamen ethanolic extract has higher concentration of in vitro and in cellulo antioxidant flavonoids than other floral components. Stamen ethanolic extract showed the highest protective effect against reactive oxygen/nitrogen species formation, as confirmed by cellular antioxidant assay using a yeast model. The evaluation of potential skin anti-aging action showed that the stamen extract has higher potential to inhibit tyrosinase and collagenase compared with its whole flower. These current findings are the first report to suggest the possibility to employ N. nucifera stamen ethanolic extract as a tyrosinase and collagenase inhibitor in cosmetic applications, as well as the utility of the current separation method.

A spectrum-effect based method for screening antibacterial constituents in Niuhuang Shangqing Pill using comprehensive two-dimensional liquid chromatography.

To investigate and screen the active antibacterial constituents of Niuhuang Shangqing Pill (NSP), the current study developed a two-dimensional liquid chromatography (2DLC) method combining microcalorimetry technique. 60% ethanol extracts from 10 batches of different commercial NSP samples were analyzed and their chemical fingerprint were developed by the comprehensive 2DLC system of Shimadzu Nexera X2. Anti-streptococcus pneumoniae (SP) constituents were determined by microcalorimetry. Thermal kinetic parameters of the SP thermogram affected by 60% ethanol extracts from 10 NSP samples were analyzed by principal component analysis. Spectrum-effect correlation between comprehensive 2DLC fingerprint and the antibacterial activity were analyzed by orthogonal partial least squares (OPLS) and orthogonal partial least squares discriminant analysis (OPLS-DA). Findings showed that peak X1 (unknown), X9 (aloe-emodin), X10 (baicalein), X11 (unknown), X14 (wogonin), X15 (glycyrrhizic acid) and X17 (unknown) are the relevant components that are in positive correlation with inhibitory rate. Regarding inhibitory rate, X17 is the most powerful one, followed by X14, X15, X10, X11, X1 and X9, suggesting that compound X17, wogonin, glycyrrhizic acid and baicalein are the major active antibacterial components of NSP. The current method employing 2DLC with microcalorimetry technique proposes a new insight for screening and identifying antibacterial components in complex herbal formula.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures.

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. .

Study of antifungal agent caspofungin adsorption to laboratory materials.

Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.

Composição química e atividades biológicas de extratos e frações de Guadua chacoensis (Rojas) Londoño & P.M. Peterson / Chemical composition and biological activities of extracts and fractions of Guadua chacoensis (Rojas) Londoño & P.M. Peterson

O Brasil é o país que possui a maior diversidade de bambus em todo o continente americano, com mais de 200 espécies catalogadas. Devido à alta resistência e durabilidade, essas espécies são muito utilizadas na construção civil e confecção de móveis e utensílios. No entanto, faltam estudos que investiguem a composição química e as atividades biológicas. Neste projeto foram avaliados extratos etanólicos de folhas e colmos de Guadua chacoensis (Rojas) Londoño & P.M. Peterson e frações em hexano, clorofórmio, acetato de etila e n-butanol. Também se obteve o óleo volátil, mas com um rendimento extremamente baixo (0,00079%). As frações dos extratos apresentaram teores de compostos fenólicos variando entre 1,92 e 15,80 µg EAG/mg. Esses compostos mostraram-se mais abundantes nas amostras de colmos. Em relação ao teor de flavonoides, as folhas apresentaram maior quantidade, variando entre 0,39 e 1,18 µg EQ/mg contra 0,17 a 0,34 µg EQ/mg nos colmos. Investigou-se a atividade antimicrobiana dos extratos, frações e óleo volátil frente cinco microrganismos: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans e Aspergillus brasiliensis. As amostras demonstraram potencial inibitório moderado a bom contra S. aureus e C. albicans, porém fraca para as demais espécies. Testou-se a capacidade antioxidante frente o radical DPPH e os resultados indicaram atividade antioxidante significativa, especialmente as frações acetato e butanol de colmos. As folhas apresentaram EC50 variando entre 67,5 e 124,0 µg/mL e os colmos entre 40,2 e 124 µg/mL. A inibição da enzima tirosinase, que está associada à produção de melanina, também se mostrou boa a uma concentração de 1 mg/mL, com o extrato bruto de colmos apresentando 43% de inibição, seguido pelas frações acetato (36%) e n-butanol (38%) de folhas. As análises por CG-MS detectaram pelo menos 44 compostos diferentes no óleo volátil, com vários terpenos e sesquiterpenos, e com ß-ionona sendo o componente majoritário (8,75%). As amostras de colmos e folhas apontaram grande diversidade de compostos, cerca de 20 para cada fração, onde os ácidos graxos como ácido palmítico e linoleico e seus ésteres derivados foram os mais abundantes. A análise dos perfis cromatográficos por CCD e CLAE revelaram a presença de ácido p-cumárico nos colmos de G. chacoensis. Esse composto tem relevante atividade antioxidante e de inibição da tirosinase. Também foi possível identificar a quercetagetina-7-O-glicosídeo, uma flavona glicosilada, com propriedades anti-inflamatorias e antidiabéticas. Desta forma, constatou-se que G. chacoensis apresenta grande diversidade de metabólitos secundários com atividades biológicas relevantes, como atividade antioxidante e clareadora, abrindo caminho para investigações mais profundas de suas aplicações, especialmente no segmento de cosméticos e produtos naturais

Brazil is the country with the greatest diversity of bamboo in the entire American continent, with more than 200 species catalogued. Due to their high resistance and durability, they are widely used in home construction and manufacture of furniture and utensils. However, studies investigating chemical composition and biological activities are absent. In this project, ethanol extracts from leaves and stems of Guadua chacoensis (Rojas) Londoño & P.M. Peterson and fractions in hexane, chloroform, ethyl acetate and n-butanol were evaluated. Volatile oil was also obtained, but with an extremely low yield (0.00079%). The fractions of the extracts presented contents of phenolic compounds varying between 1.92 and 15.80 µg GAE/mg. These compounds were more abundant in culm samples. In relation to the flavonoid content, leaves showed a greater amount, varying between 0.39 and 1.18 µg QE/mg against 0.17 to 0.34 µg QE/mg in culms. The antimicrobial activity of extracts, fractions and volatile oil were investigated against five microorganisms: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus brasiliensis. The samples showed moderate to good inhibitory potential against S. aureus and C. albicans, but weak for the other species. The antioxidant capacity was tested against the DPPH radical and the results indicated significant antioxidant activity, especially acetate and butanol culm fractions. The leaves presented EC50 varying between 67.5 and 124.0 µg/mL and culms between 40.2 and 124 µg/mL. The inhibition of the enzyme tyrosinase, which is associated with the production of melanin, was also shown to be good at a concentration of 1 mg/mL, with the raw culm extract showing 43% inhibition, followed by acetate (36%) and n-butanol (38%) fractions of leaves. CG-MS analysis detected at least 44 different compounds in volatile oil, with several terpenes and sesquiterpenes, and with ß-ionone being the major component (8.75%). Culm and leaf samples showed great diversity of compounds, about 20 for each fraction, where fatty acids such as palmitic and linoleic acid and their derivative esters were the most abundant. The analysis of the chromatographic profiles by TLC and HPLC revealed the presence of p-coumaric acid in culms of G. chacoensis. This compound has relevant antioxidant and tyrosinase inhibiting activity. It was also possible to identify quercetagetine-7-O-glucoside, a glycosylated flavone, with anti-inflammatory and anti-diabetic properties. Thus, it was found that G. chacoensis presents a great diversity of secondary metabolites with relevant biological activities, such as antioxidant and whitening activity, opening ways for deeper investigations of its applications, especially in the segment of cosmetics and natural products

A novel, sensitive, and widely accessible besifloxacin quantification method by HPLC-fluorescence: Application to an ocular pharmacokinetic study.

Besifloxacin has been embraced for the treatment of ocular bacterial infections. While LC-MS/MS has been used in investigating BSF pharmacokinetics, those costly instruments are not universally available and have complicated requirements for operation and maintenance. Additionally, pharmacokinetics of besifloxacin in dose-intense regimens are still unknown. Herein, a new quantification method was developed employing the widely accessible HPLC with fluorescence detection and applied to an ocular pharmacokinetic study with an intense regimen. Biosamples were pre-treated using protein precipitation. Chromatographic separation was achieved on a C18 column using mobile phase of 0.1% trifluoroacetic acid and acetonitrile. To address the weak fluorescence issue of besifloxacin, effects of detection parameters, elution pattern, pH of mobile phase, and reconstitution solvents were investigated. The method was fully validated per US-FDA guidelines and demonstrated precision (<13%), accuracy (91-112%), lower limit of quantification (5 ng/mL), linearity over clinically relevant concentrations (R2 > 0.999), matrix-effects (93-105%), recoveries (95-106%), and excellent selectivity. The method showed agreement with agar disk diffusion assays for in vitro screening and comparable in vivo performance to LC-MS/MS (Deming Regression, y = 1.010x + 0.123, r = 0.997; Bland-Altman analysis, mean difference was -6.3%; n = 21). Pharmacokinetic parameters suggested superior surface-retentive properties of besifloxacin. Maximum concentrations were 1412 ± 1910 and 0.15 ± 0.12 µg/mL; area under the curve was 1,637 and 1.08 µg·h/g; and half-life was 4.9 and 4.1 h; and pharmacokinetic-to-pharmacodynamic ratios were ≥ 409 and ≤ 17.8 against ocular pathogens in tears and aqueous humor, respectively. This readily available method is sensitive for biosamples and practical for routine use, facilitating besifloxacin therapy development.

Regulated and Non-Regulated Mycotoxin Detection in Cereal Matrices Using an Ultra-High-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UHPLC-HRMS) Method.

Cereals represent a widely consumed food commodity that might be contaminated by mycotoxins, resulting not only in potential consumer health risks upon dietary exposure but also significant financial losses due to contaminated batch disposal. Thus, continuous improvement of the performance characteristics of methods to enable an effective monitoring of such contaminants in food supply is highly needed. In this study, an ultra-high-performance liquid chromatography coupled to a hybrid quadrupole orbitrap mass analyzer (UHPLC-q-Orbitrap MS) method was optimized and validated in wheat, maize and rye flour matrices. Nineteen analytes were monitored, including both regulated mycotoxins, e.g., ochratoxin A (OTA) or deoxynivalenol (DON), and non-regulated mycotoxins, such as ergot alkaloids (EAs), which are analytes that are expected to be regulated soon in the EU. Low limits of quantification (LOQ) at the part per trillion level were achieved as well as wide linear ranges (four orders of magnitude) and recovery rates within the 68-104% range. Overall, the developed method attained fit-for-purpose results and it highlights the applicability of high-resolution mass spectrometry (HRMS) detection in mycotoxin food analysis.

Ultra-high-performance liquid chromatography high-resolution mass spectrometry variants for metabolomics research.

Ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) variants currently represent the best tools to tackle the challenges of complexity and lack of comprehensive coverage of the metabolome. UHPLC offers flexible and efficient separation coupled with high-sensitivity detection via HRMS, allowing for the detection and identification of a broad range of metabolites. Here we discuss current common strategies for UHPLC-HRMS-based metabolomics, with a focus on expanding metabolome coverage.

A New Parallel High-Pressure Packing System Enables Rapid Multiplexed Production of Capillary Columns.

Reversed-phase HPLC is the most commonly applied peptide-separation technique in MS-based proteomics. Particle-packed capillary columns are predominantly used in nanoflow HPLC systems. Despite being the broadly applied standard for many years, capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column packing with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as the driving fluid, our system replaces the traditional setup of helium-pressured packing bombs. By using 10× multiplexing, we have reduced the production time to just under 2 min for several 50 cm columns with 1.9-µm particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters and lengths packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station.

Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC-ELSD to reveal accumulation differences of sugar and lipid.

OBJECTIVES: To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes between carbohydrate and lipid in fermentation process. RESULTS: The extraction of mycelia with HCl following a photometric phenol-sulphuric acid reaction was identified as an optimal method for total carbohydrate analysis in Mortierella alpina, which the extraction efficiency performed 1.1-3.6 fold than other five methods. The total carbohydrate content increased from initial 19.26 to 25.86% during early fermentation process and declined gradually thereafter, while the fatty acid was increasing from 8.47 to 31.03%. For separation and qualitative estimation of intracellular saccharides, the acetonitrile/water freeze-thaw method for extraction and Sugar-Pak I column for separation proved to be possible. With the glucose rapidly decreasing at the beginning of growth, the trehalose accumulated rapidly from 1.63 to 5.04% and then decreased slightly but maintain above 4% of dry biomass. CONCLUSIONS: This work established comprehensive carbohydrate extraction and analysis methods of Mortierella alpina and identified the main saccharide in fermentation process which indicated that the accumulation of fatty acids was related to the change of intracellular carbohydrate content.

A novel quality by design approach for development and validation of a green reversed-phase HPLC method with fluorescence detection for the simultaneous determination of lesinurad, febuxostat, and diflunisal: Application to human plasma.

A novel and eco-friendly reversed-phase HPLC method with fluorescence detection was developed for simultaneous estimation of two co-administered antigout drugs (lesinurad and febuxostat) with diflunisal as a nonsteroidal anti-inflammatory drug. Unlike routine methodology, the developed method was optimized using analytical quality by design approach. A full factorial design was applied to optimize the effect of variable factors on chromatographic responses. The chromatographic separation was performed using isocratic elution on the Hypersil BDS C18 column at 40°C. The mobile phase consisted of acetonitrile:potassium phosphate buffer (30.0 mM; pH 5.5, 32.2:67.8% v/v) pumped at a flow rate of 1.0 mL/min and injection volume of 20.0 µL was employed. The proposed method was able to separate the ternary mixture in <10 min. The calibration curves of diflunisal, lesinurad, and febuxostat were linear over concentration ranges of 50.0-500.0, 50.0-700.0, and 20.0-700.0 ng/mL, respectively. Recovery percentages ranging from 98.1 to 101.3% with % relative standard deviation of <2% were obtained upon spiking to human plasma samples, indicating high bioanalytical applicability. Furthermore, the method was found to be excellent green when it was assessed according to Green Analytical Procedure Index and analytical Eco-Scale guidelines.

A new method of extracting polyphenols from honey using a biosorbent compared to the commercial resin amberlite XAD2.

A new extraction method of polyphenols from honey using a biodegradable resin was developed and compared with the common commercial resin amberlite XAD2. For this purpose, three honey samples of Algerian origin were selected for the different physicochemical and biochemical parameters study. After extraction of the target compounds by both resins, the polyphenol content was determined, the antioxidant activity was tested, and liquid chromatography-mass spectrometry analyses were performed for identification and quantification. The results showed that physicochemical and biochemical parameters meet the norms of the International Honey Commission, and the H1 sample seemed to be of high quality. The optimal conditions of extraction by biodegradable resin were a pH of 3, an adsorption dose of 40 g/L, a contact time of 50 min, an extraction temperature of 60°C, and no stirring. The regeneration and reuse number of both resins was three cycles. The polyphenol contents demonstrated a higher extraction efficiency of biosorbent than of XAD2, especially in H1. Liquid chromatography-mass spectrometry analyses allowed for the identification and quantification of 15 compounds in the different honey samples extracted using both resins and the most abundant compound was 3,4,5-trimethoxybenzoic acid. In addition, the biosorbent extracts showed stronger antioxidant activities than the XAD2 extracts.

Polyaniline/titanium dioxide nanorods functionalized carbon fibers for in-tube solid-phase microextraction of phthalate esters prior to high performance liquid chromatography-diode array detection.

To improve extraction performance of carbon fibers (CFs) towards phthalate esters (PAEs), titanium dioxide (TiO2) nanorods array was in-situ grown on the surface of CFs, then polyaniline (PANI) was used to modify it. PANI/TiO2 nanorods-CFs were placed into a polyetheretherketone tube for solid-phase microextraction (SPME). Combining the tube to high performance liquid chromatography (HPLC), it was evaluated and displayed good extraction performance for several PAEs. Compared with bare CFs, TiO2 nanorods and PANI, PANI/TiO2 nanorods presented best performance, attributed to the unique advantages between high surface area of TiO2 nanorods and multiple adsorption interactions (like π-π stacking, hydrogen bond) of PANI. After the optimization of the important factors (sampling volume, sampling rate, sample pH, concentrations of organic solvent and salt in sample, and desorption time), the online in-tube SPME-HPLC method was established. It provided low limits of detection (0.01-0.05 µg L-1) and wide linear ranges (0.03-30, 0.10-30, 0.17-30 µg L-1) with correlation coefficients larger than 0.9991. The relative standard deviations (n=6) between intra-day and inter-day tests were in the ranges of 3.5-10.3% and 4.7-13.9%, respectively. The method was successfully used to determine seven PAEs in real water samples. Besides of satisfactory durability, the material also exhibited superior extraction performance than some materials.

Isolation of Extracellular Vesicles (EVs) Using Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC).

Extracellular vesicles (EVs), are membrane-bound nanoparticles of biological origin. These signature molecules of health and disease have raised remarkable attention of the biomedical arena due to its potential diagnostic and therapeutic applicability. Among the many different techniques available for EV isolation, size-exclusion chromatography (SEC) is widely accepted.In this chapter, we present a protocol of size-exclusion high-performance liquid chromatography (SE-HPLC) as a method of EV isolation. This method can be adapted as a low cost but a reliable and scalable method of EV isolation in those laboratories having access to the HPLC systems.

Fundamental to achieving fast separations with high efficiency: A review of chromatography with superficially porous particles.

Types of particles have been fundamental to LC separation technology for many years. Originally, LC columns were packed with large-diameter (>100 µm) calcium carbonate, silica gel, or alumina particles that prohibited fast mobile-phase speeds because of the slow diffusion of sample molecules inside deep pores. During the birth of HPLC in the 1960s, superficially porous particles (SPP, ≥30 µm) were developed as the first high-speed stationary-phase support structures commercialized, which permitted faster mobile-phase flowrates due to the fast movement of sample molecules in/out of the thin shells. These initial SPPs were displaced by smaller totally porous particles (TPP) in the mid-1970s. But SPP history repeated when UHPLC emerged in the 2000s. Stationary-phase support structures made from sub-3-µm SPPs were introduced to chromatographers in 2006. The initial purpose of this modern SPP was to enable chromatographers to achieve fast separations with high efficiency using conventional HPLCs. Later, the introduction of sub-2-µm SPPs with UHPLC instruments pushed the separation speed and efficiency to a very fast zone. This review aims at providing readers a comprehensive and up-to-date view on the advantages of SPP materials over TPPs historically and theoretically from the material science angle.

Comparison of QuEChERS and "dilute and shoot" extraction methods for multi-mycotoxin analysis of samples from button mushroom (Agaricus bisporus) cultivation.

Several components of mushroom compost (wheat straw, chicken manure) can be contaminated with mycotoxins posing food health risks to mushroom consumers. To assess the relevance of such contaminations high-throughput analytical methods are needed. In this study, two sample preparation approaches, dilute & shoot (D&S) and modified citrate buffered Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) were compared in terms of extraction efficiency and matrix effect in case of 13 mycotoxins in complex matrices-wheat straw, the growing media and button mushrooms (Agaricus bisporus)-of mushroom cultivation using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). D&S method resulted in recoveries of LB medium, button mushroom and compost for ≥60% in case of all investigated mycotoxins except for DON-3G. However, using modified citrate buffered QuEChERS with 2% acidification of the extraction solvent showed the complete loss of strongly polar DON-3G and fumonisin B1 (FB1). The investigated matrices had suppressive effect on ionization in all target mycotoxins except for FB1. Regarding the use of isotopologues to compensate matrix effect, even U-[13C15]-DON and U-[13C24]-T-2 can also be used to quantify their related metabolites in the studied matrices, using internal standard method.

Power function setting in charged aerosol detection for the linearization of detector response - optimization strategies and their application.

Charged aerosol detection (CAD) is an universal technique in liquid chromatography that is increasingly used for the quality control of drugs. Consequently, it has found its way into compendial monographs promoted by its simple and robust application. However, the response of CAD is inherently nonlinear due to its principle of function. Thus, easy and rapid linearization procedures, in particular regarding compendial applications, are highly desirable. One effective approach to linearize the detector's signal makes use of the built-in power function value (PFV) setting of the instrument. The PFV is basically a multiplication factor to the power law exponent of the equation describing the CAD's response, thereby altering the detector's signal output to optimize the quasi-linear range of the response curve. The experimental optimization of the PFV for a series of analytes is a time-consuming process, limiting the practicability of this approach. Here, two independent approaches for the determination of the optimal PFV based on an empirical model and a mathematical transformation in each case, are evaluated. Both approaches can be utilized to predict the optimal PFV for each analyte solely based on the experimental results of a series of calibration standards obtained at a single PFV. The approaches were applied to the HPLC-UV-CAD impurity analysis of the drug gabapentin to improve the observed nonlinear response of the impurities in the range of interest. The predicted optimal PFV of both approaches were in good agreement with the experimentally obtained optimal PFV of the analytes. As a result, the accuracy of the method was significantly improved when using the optimal PFV (90 - 105% versus 81 - 115% recovery rate for quantitation by either single-point calibration or linear regression) for the majority of the analytes. The final method with a PFV adjusted to 1.30 was validated with respect to ICH guideline Q2(R1).

Soil dissipation of sugarcane billet seed treatment fungicides and insecticide using QuEChERS and HPLC.

Chemical treatment of sugarcane seed with fungicides and insecticides prior to planting increases yields of cane and sugar for the perennial, annually harvested crop. However, the fate of the applied chemicals is unknown. Therefore, the purpose of this study was to measure the aerobic dissipation of selected billet seed treatment chemicals in a mineral sugarcane soil from Louisiana. Soil samples from the surface 15 cm were treated with either thiamethoxam, azoxystrobin, fluxapyroxad, propiconazole, or pyraclostrobin and monitored over 100 days under laboratory conditions. Insecticide and fungicide levels were determined by high performance liquid chromatography. Dissipation data were fitted to four kinetic models: simple first-order (SFO), first order multi-compartment (FOMC), double-first order in parallel (DFOP), and hockey-stick (HS). The dissipation half-life (DT50) of thiamethoxam, azoxystrobin, fluxapyroxad, propiconazole, or pyraclostrobin were 275, 100, 144, 74, and 39 d, respectively. Overall, the DT50 for the pesticides in the study indicated medium to long persistence in soil under the conditions of the experiment. This is the first report for several of these pesticides related to the aerobic dissipation in soils used to grow sugarcane.

Combining Field-Enabled Capillary Vibrating Sharp-Edge Spray Ionization with Microflow Liquid Chromatography and Mass Spectrometry to Enhance 'Omics Analyses.

Field-enabled capillary vibrating sharp-edge spray ionization (cVSSI) has been combined with high-flow liquid chromatography (LC) and mass spectrometry (MS) to establish current ionization capabilities for metabolomics and proteomics investigations. Comparisons are made between experiments employing cVSSI and a heated electrospray ionization probe representing the state-of-the-art in microflow LC-MS methods for 'omics studies. For metabolomics standards, cVSSI is shown to provide an ionization enhancement by factors of 4 ± 2 for both negative and positive ion mode analyses. For chymotryptic peptides, cVSSI is shown to provide an ionization enhancement by factors of 5 ± 2 and 2 ± 1 for negative and positive ion mode analyses, respectively. Slightly broader high-performance liquid chromatography peaks are observed in the cVSSI datasets, and several studies suggest that this results from a slightly decreased post-split flow rate. This may result from partial obstruction of the pulled-tip emitter over time. Such a challenge can be remedied with the use of LC pumps that operate in the 10 to 100 µL·min-1 flow regime. At this early stage, the proof-of-principle studies already show ion signal advantages over state-of-the-art electrospray ionization (ESI) for a wide variety of analytes in both positive and negative ion mode. Overall, this represents a ∼20-50-fold improvement over the first demonstration of LC-MS analyses by voltage-free cVSSI. Separate comparisons of the ion abundances of compounds eluting under identical solvent conditions reveal ionization efficiency differences between cVSSI and ESI and may suggest varied contributions to ionization from different physicochemical properties of the compounds. Future investigations of parameters that could further increase ionization gains in negative and positive ion mode analyses with the use of cVSSI are briefly presented.

Preparation of value-added metal-organic frameworks for high-performance liquid chromatography. Towards green chromatographic columns.

This work applies the concepts of green chemistry, where polyethylene terephthalate (PET) bottles were used as the acid-dicarboxylic linker source for the synthesis of MIL-53(Al) metal organic frameworks (MOFs) and then used as a stationary phase for the separation of various solutes and compared with MIL-53(Al) synthesized from traditional terephthalic acid. Both synthesized MIL-53(Al) MOFs were characterized by scanning electron microscopy (SEM), FT-IR, X-ray diffraction (XRD), thermogravimetric analysis (TGA), and specific surface area analysis. Eight groups of standard analytes in addition to real samples were tested to evaluate the separation performance of the MIL-53(Al) packed columns in HPLC under various chromatographic conditions. Based on elution order of the studied compounds and the effects of mobile phase composition, the working mechanism was reversed phase mode in the presence of size-exclusion effects for large molecules, which exceeded the dynamic diameter of MIL-53(Al) (~7.6 Å). The effects of stationary phase sieving, mobile phase flow rate and composition, injected sample mass, and temperature were investigated relative to the chromatographic behavior of MIL-53(Al). MIL-53(Al) particle sieving before packing reduced peak broadening and significantly enhanced the chromatographic performance of the prepared columns up to 2.26 times relative to the number of theoretical plates. The MIL-53(Al) packed columns offered high-resolution separation for all studied mixtures with Rs >2 and good stability and long-term durability. At optimal conditions, the prepared columns exhibited efficiencies between 5600-63200 plates m-1. Higher efficiencies were observed for alkylbenzenes and polyaromatic hydrocarbons as the organic linker in the MIL-53(Al) structure, which improved retention and separation of aromatics through π-π interactions. Thermodynamic parameters including ΔH, ΔS, and ΔG for the transfer of analyte from the mobile phase to the MIL-53(Al) stationary phase were studied. Compared with previously cited MOFs packed columns, the present MIL-53(Al) columns gave comparable selectivity and much better efficiency for most of the studied chemicals at optimum conditions, indicating the feasibility of MIL-53(Al) as a stationary phase for HPLC applications.