Repeatability Of Retention Times Research Articles

Determination of Total Apigenin in Herbs by Micellar Electrokinetic Chromatography with UV Detection.

Apigenin is a naturally occurring plant flavone that exhibits strong antioxidant, anti-inflammatory, and antitumor properties. A MEKC-UV based method was developed for the determination of total apigenin in selected herbs. Application of pseudostationary phase in the form of SDS micelles resulted in great repeatability of retention times and peak areas. A buffer solution consisting of 30 mmol/L sodium borate (pH 10.2), 10% acetonitrile, and 10 mmol/L sodium dodecyl sulfate was found to be the most suitable BGE for the separation. The method was validated and calibrated for total apigenin in the range of 1.0–100 μmol/L (R 2 = 0.9994). The limits of detection and quantification were 0.48 μmol/L and 0.92 μmol/L, respectively. This precise and robust method was successfully applied to the analysis of plant samples for total apigenin content.

Determination of cathinones and other stimulant, psychedelic, and dissociative designer drugs in real hair samples.

The detection of new psychoactive substances (NPS) in hair proved to provide insight into their current diffusion among the population and the social characteristics of these synthetic drugs' users. Therefore, a UHPLC-MS/MS method was developed in order to determine 31 stimulant and psychedelic substituted phenethylamines, and dissociative drugs in hair samples. The method proved to be simple, fast, specific, and sensitive. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method showed optimal linearity in the interval 10-1000 pg/mg, with correlation coefficient values varying between 0.9981 and 0.9997. Quantitation limits ranged from 1.8 pg/mg for 4-methoxyphencyclidine (4-MeO-PCP) up to 35 pg/mg for 6-(2-aminopropyl)benzofuran (6-APB). The method was applied to (i) 23 real samples taken from proven MDMA and ketamine abusers and (ii) 54 real hair samples which had been previously tested negative during regular drug screening in driver's license recovery. Six samples tested positive for at least one target analyte. Methoxetamine (MXE) was found in three cases (range of concentration 7.7-27 pg/mg); mephedrone (4-MMC) was found in two cases (50-59 pg/mg) while one sample tested positive for methylone at 28 pg/mg. Other positive findings included 4-methylethcathinone (4-MEC), alpha-pyrrolidinovalerophenone (α-PVP), 4-fluoroamphetamine (4-FA), 3,4-methylenedioxypyrovalerone (MDPV), and diphenidine. The present study confirms the increasing diffusion of new designer drugs with enhanced stimulant activity among the target population of poly-abuse consumers.

Piston-cylinder based micro liquid–liquid extraction with GC–qMS for trace analysis of targeted chlorinated organic compounds in water

A fast and reliable approach for the measurement of sub parts-per-billion levels of targeted chlorinated compounds like tetrachloroethane, hexachloroethane, hexachlorobutadiene, pentachlorobenzene, and hexachlorobenzene in various water matrices such as waste water is described. The method employed a novel piston-cylinder-based micro liquid–liquid extraction technique using hexane as an extraction solvent. The device, known commercially as the MIXXOR, substantially accelerates extraction time by a factor of more than 100 times and reduces solvent consumption by a factor of 25 times when compared with the solvent extraction technique using wrist-action mechanical agitation. A recently introduced 6% cyanopropylphenyl –94% dimethylpolysiloxane capillary column offering a high degree of inertness was used for the separation of the analytes. A quadrupole mass spectrometer equipped with a triple-axis detector was also employed to enhance the instrument detection limit. With this technique, a complete separation for the analytes in water can be conducted in less than 10 min using a three-port SilFlow planar microfluidic device for back-flushing. Repeatability of retention times for all compounds were found to be less than 0.04% (n = 10). The compounds cited can be analyzed from 1 ng/mL to 10 μg/mL, with a detection limit and correlation coefficient of at least 0.5 ng/mL and 0.999, respectively. A relative precision of less than 1.2% relative standard deviation (RSD) (n = 20) at the 50 ng/mL level, with analyte recovery of greater than 99% (n = 3) from 10 ng/mL to 10 μg/mL, was obtained.

Back-flushing and heart cut capillary gas chromatography using planar microfluidic Deans’ switching for the separation of benzene and alkylbenzenes in industrial samples

Planar microfluidic devices coupled with modern electronic pressure control have allowed gas chromatography (GC) practitioners to easily manipulate chromatographic systems to achieve heart cut and back-flushing configurations. These planar microfluidic devices have enhanced the connectivity between different components of GC instrumentation and have improved the inertness and minimised system dead volumes compared to classical chromatographic unions and valves. In the present contribution the setup and configuration of two multidimensional GC (MDGC) platforms is described for achieving the separation and quantification of trace level target C6–C8 alkylbenzenes in styrene monomer and Isoparaffin™ solvents, using flame ionisation detection (FID). The performance of these MDGC platforms indicated excellent retention time (0.2% relative standard deviation, RSD) and peak area repeatability (1% RSD) for all analytes of interest. The limit of detection (LOD) was 0.8mgkg−1 for benzene in styrene monomer, and 2.4–2.8mgkg−1 for C6–C8 alkylbenzenes such as benzene, toluene, ethylbenzene and xylene in Isoparaffin™ solvent.

Miniaturised medium pressure capillary liquid chromatography system with flexible open platform design using off-the-shelf microfluidic components

Trends towards portable analytical instrumentation of the last decades have not been equally reflected in developments of portable liquid chromatography (LC) instrumentation for rapid on-site measurements. A miniaturised medium pressure capillary LC (MPLC) system with gradient elution capability has been designed based on a flexible modular microfluidic system using primarily off-the-shelf low cost components to ensure wide accessibility to other analysts. The microfluidic platform was assembled on a breadboard and contained microsyringe pumps and switch valves, complemented with an injection valve and on-capillary detectors, all controlled by a PC. Four miniaturised microsyringe pumps, with 5, 20 and 100 μL syringe volume options, formed the basis of the pumping system. Two pairs of pumps were used for each mobile phase to create gradient elution capability. The two microsyringe pumps in each pairs were linked by two electrically operated microfluidic switching valves and both pairs of pumps were connected through a zero void volume cross-connector, thus providing a low hold-up volume for gradient formation. Sample was injected by a 20 nL nano-LC sampling valve, directly connected to a 18 cm long 100 μm i.d. Chromolith CapRod RP-18 monolithic capillary column. On-capillary LED-based UV–vis photometric detection was conducted through a piece of equal diameter fused silica capillary connected after the column. The performance of the portable LC system was evaluated theoretically and experimentally, including the maximum operating pressure, gradient mixing performance, and the performance of the detectors. The 5 μL microsyringe pump offered the best performance, with typical maximum operating pressures up to 11.4 ± 0.4 MPa (water) and gradient pumping repeatability of between 4 and 9% for gradients between 0.10% s−1 and 0.33% s−1. Test analytes of charged and uncharged dyes and pharmaceuticals of varying hydrophobicity showed typical RSD values of 0.7–1.4% and 3.3–4.8% in isocratic mode and 1.2–4.6% and 3.2–6.4% in gradient mode, respectively for retention time and peak area repeatability.

Separation Study of Eight Isoflavones by MEKC with Different Surfactants

Isoflavones are a very important group of natural products. This study investigated the separation of eight isoflavones, namely ononin, daidzin, genistin, biochanin A, formononetin, puerarin, genistein, and daidzein, from pueraria by micellar electrokinetic chromatography (MEKC) with different surfactants. The following micellar systems of MEKC were systematically compared for the analysis of these isoflavones: (1) a single surfactant comprising the anionic surfactant sodium dodecyl sulfate (SDS), the cationic surfactant hexadecyltrimethylammonium bromide, the neutral surfactant polyoxyethylene sorbitan monolaurate (Tween 20), and the ionic liquid-type surfactant (also a cationic surfactant) 1-dodecyl-3-methylimidazolium tetrafluoroborate (C12MIMBF4); (2) different single surfactants with 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF4) as an additive (modifier); and (3) mixed micelles of SDS + Tween 20 and C12MIMBF4 + Tween 20. Both SDS with BMImBF4 as additive and mixed micelles of SDS + Tween 20 had the highest separation efficiency for the eight investigated compounds. Furthermore, the SDS with BMImBF4 as additive was more stable (good repeatability of retention time and peak shape of analytes) than mixed micelles of SDS + Tween 20, which may be the result of a stabilizing effect of BMImBF4. Therefore, the final analytical conditions were 15 mM SDS added with 50 mM BMImBF4 in 30 mM sodium tetraborate (STB, pH 9.5) as running buffer; applied voltage, 20 kV; injection, 50 mbar for 5 s; cartridge temperature, 25 °C; compounds were detected at 260 nm. The developed method was fully validated (limit of detection, limit of quantification, intraday precision, inter-day precision, and recovery) and successfully applied to determine the eight analytes in three Radix Puerariae samples. The present study indicated that SDS with ionic liquids as additive in MEKC was suitable for the analysis of isoflavones.

Capillary electrochromatography-mass spectrometry for the determination of 5-nitroimidazole antibiotics in urine samples.

The separation of eight antibiotics belonging to 5-nitroimidazole family was carried out by means of CEC coupled with MS. Preliminary experiments were carried out with ultraviolet detection in order to select the proper stationary and mobile phase. Among the different stationary phases studied (namely Lichrospher C18, 5 μm particle size; Cogent(TM) Bidentate C18, 4.2 μm; Pinnacle II™ Phenyl, 3 μm; Pinnacle II™ Cyano, 3 μm), Cogent™ Bidentate C18 (4.2 μm) gave the best performance. For CEC-MS coupling, a laboratory assembled liquid-junction-nano-spray interface was used. In order to achieve a good sensitivity, special attention was paid to both optimization of the sheath liquid composition as well as selection of the injection mode. Under optimized CEC-ESI-MS conditions, the separation was accomplished within 22 min by using a column packed with a mixture of Bidentate C18:Lichrospher Silica-60 (5 μm) 3:1 w/w, an inlet pressure of 11 bar, a voltage of 15 kV, and a mobile phase composed by 45:10:45 v/v/v ACN/MeOH/water containing ammonium acetate (5 mM pH 5). A combined hydrodynamic and electrokinetic injection of 8 bar, 15 kV, and 96 s was adopted. The method was validated in terms of repeatability and intermediate precision of retention times and peak areas, linearity, and LODs and LOQs. RSDs values were <2.9% for retention times and <16.1% for peak areas in both intraday and interday experiments. LOQ values were between 0.09 and 0.42 μg/mL for all compounds. Finally, the method was applied to the determination of three most employed 5-nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in spiked urine samples, subjected to a SPE procedure. Recovery values in the 67-103% range were obtained. Furthermore, for the selected antibiotics, CEC-MS(2) spectra were obtained providing the unambiguous confirmation of these drugs in urine samples.

Stage-frit: A straightforward sub-2 μm nano-liquid chromatography column fabrication for proteomic analysis

In this work we demonstrated a facile method for the fabrication of C18 coordination polymer gel in a capillary, called stage-frit, which was efficiently applied to pack sub-2 μm C18 beads into the capillary by a high pressure bomb for the online separation of proteolytic peptides. The back pressure of the column with 10 cm × 75 μm i.d. is regularly lower than 170 bar at a flow rate of 300 nl/min, which could be operated on a common nanoLC system instead of nanoUPLC system due to the good permeability, low back pressure and high mechanical stress of the frit that will totally reduce the cost for the purchase of instrument. The stage-frit allows long-term continuous flow of the solvent and no significant beads loss or pressure instability was observed during the period. The repeatability of retention time for fifteen BSA tryptic peaks was found to be less than 1.08% (RSD) in six time nanoLC-ESI-MS/MS experiments. The average full width at half maximum (FWHM) of peptide peaks is 5.87 s. The sub-2 μm stage-frit nanoLC column showed better sensitivity than the commercial available for large scale proteomic analysis of total tissue proteins from human spleen. The number of identified peptides is approximately 0.4-fold and 0.2-fold higher than that obtained by utilizing commercial columns packed with 3 μm and 1.8 μm C18 materials, respectively. In the field of analytical chemistry, particularly the use of nanoLC systems, stage-frit nanoLC column offers a great potential for the separation of complex mixtures.

Blood Orange Anthocyanins in Fruit Beverages: How the Commercial Shelf Life Reflects the Quality Parameter

Pigmented oranges are a high value typical product, used for the production of healthy juice-based drinks due to the peculiar presence of anthocyanins as dietary antioxidants. A qualitative HPLC/PDA/MS analytical method for verifying presence/absence of blood orange anthocyanins was validated in accordance with Eurachem guide in terms of accordance (100%), specificity/selectivity, repeatability of retention time (CV &lt; 0.5%), limit of detection (0.36 mg/L) and limit of decision (0.41 mg/L). Fifty commercial red orange juice beverages were collected on the market and analyzed to assess the occurrence of blood orange anthocyanins. The results showed that only 60% of analyzed products have a minimum content of anthocyanins of blood orange, guaranteeing the specifications of the product they publicize and sell until the expiration date. Therefore, it is possible to envisage a gradual degradation of the specific parameter (anthocyanins) or willful misconduct of producers (non-use of blood orange juice). In both cases, the consumer buys a product with high added value without such value being guaranteed. This information will be useful to revise the expiration date of these products, the percentage of blood orange juice used in the preparation and/or the storage conditions of the products.

Solid-phase extraction and nanoflow liquid chromatography-nanoelectrospray ionization mass spectrometry for improved global urine metabolomics.

Global urine metabolomics is a rapidly expanding field with the potential to discover biomarkers of disease and exposure. To date, most methods focus on rapid sample preparation, using neat or diluted urine, together with high-throughput analyses, and are poorly suited for detection of low abundance metabolites present in urine samples. In this study, novel methods have been developed to analyze urine by splitless nanoflowUHPLC-nanoESI-TOFMS (nUHPLC-nESI-TOFMS) after preconcentration by solid-phase extraction (SPE), thus enabling significant improvements in analytical sensitivity and coverage of the urinary metabolome. In initial work, urine samples were extracted by both anion and cation exchange mixed-mode polymeric SPE cartridges and qualitatively compared with those using conventional sample preparations using UHPLC-ESI-TOFMS analyses. Compared with neat or diluted urine samples, SPE concentration of urine resulted in detection of additional metabolites including bile acids, lipids, pharmaceuticals, and markers of lifestyle, with little loss of other components of the metabolome. Analyses of SPE preparations by nUHPLC-nESI-TOFMS revealed excellent retention time repeatability with <1% coefficient of variation (CV) for 96% of analyzed peaks. The repeatability of the MS response was <30% CV for >79% of MS features in both negative and positive nESI modes. Compared with UHPLC-ESI-TOFMS, analysis by the nanoplatform enabled detection of signaling molecules important in disease processes including sex steroids, glucocorticoids, eicosanoids, and neurotransmitter metabolites. The significant improvement in sensitivity arising from use of splitless nUHPLC-nESI-TOFMS analyses of SPE-concentrated samples represents a step change in coverage of the urinary metabolome, thereby increasing the potential for biomarker discovery.

Nutrition care and exercise: An effect on muscle and fat metabolism in dialysed and transplant patients with obesity

s / Nutrition 30 (2014) 1222–1224 1224 response in the concentration range from LOQ to 55 ng for CH and LOQ to 114 ng for 7-OCH. The response factors (in relation to 5-alpha-cholestane) were 0.206 and 0.142 for CH and for 7-OCH, respectively. System and method precisions were sufficient, repeatability of retention time was less than 1 %, repeatability of injection was less than 15%; the repeatability of the whole procedure for cholesterol was 15.8%. Conclusion: The method of SPE separation of cholesterol and its oxidized products form fat matrices followed by GC-MS was adopted and optimized and working characteristics were evaluated. The method is precise, linear and sufficiently sensitive to determine oxysterols in both food and biological materials. Our future work will be focus on applying this method to the evaluation of oxysterols in fish, meat and vegetable dishes created under different types of culinary conditions (frying, baking, roasting). NUTRITION CARE AND EXERCISE: AN EFFECT ON MUSCLE AND FAT METABOLISM IN DIALYSED AND TRANSPLANT PATIENTS WITH OBESITY V. Teplan , I. Kr alov a-Lesn a , A. Mahrov a , M. H ajek , J. Racek , M. Stollov a 1 Dept of Nephrol, Transplant Centre, Inst Clin Exp Medicine, Prague Cardiovascular research centre, Prague, Czech Republic 3 Faculty of Physical Educ and Sport, Charles Univ Prague, Czech Republic Dept of Magnetic Resonance Imaging,Ist Clin Exper Medicine, Prague Dept of Biochem, Charles Univ Pilsen, Czech Republic Rationale: Vascular lesions during long-term dialysis pose a major limitation for organ transplantation. Endothelial progenitor cells (EPC) play important role in the integrity of vascular endothelium and may be integrated into endothelial cells. Recently, there have been studies documenting the beneficial effect of regular physical activity and nutrition on EPC count and onmuscle and fat metabolism in dialyzed and renal transplant patients. The aim of prospective randomized study was to assess the relationship between peripheral EPC, plasma asymmetric dimethylarginine (ADMA), adipocytokines, visceral fat and fat in muscle mass examined by magnetic resonance spectroscopy (MRS) in dialysed (pre-transplant) and transplant patients before and after exercise training and nutritional management. Methods: We monitored 65 patients before (on dialysis) and 6 months after first cadaveric kidney transplantation (Group I). Intervention involved controlled exercise regimen (Rikli Jones, HRQOL SF36-Bref) and nutritional care. Control Group II consisted of 63 non-exercising transplant patients with standard diet matched in age, BMI, HLA typisation, dialysis management, history of cardiovascular disease and immunosuppressive regimen. Nutritional management in Group I consisted of modified medium protein and energy diet (0.8-1.0g/kg and 35 kcal/kg iBW/day supplemented with mixture of keto,hydroxyand L-amino acids/100mg/kg iBW/day). EPC count in peripheral blood, laboratory examination and MRS were performed before and at month 6 after kidney transplantation. Results: After six months period, significant differences were found in EPC, ADMA, adiponectin and visfatin, (ttest, ANOVA all p < 0.01) in Group I. MRS examinations confirmed reducing of extracellular muscle fat from abdominal and calf muscle area after exercise training (p<0.01).This effect was not found in patients with body/fat weight gain.Renal function was stabilized during follow-up and all patients with rejection were excluded. Conclusion: Regular exercise training and nutritional care were associated with increase of EPC and adiponectin, decrease of ADMA and reducing of fat in muscles in non-obese patients examined by MRS in Group I. This regimen may decrease the vascular risk after organ transplantation and remarkable influence patient s well-being. The study was supported by Project MZO NT 13139-3/2012 awarded by the Internal Grant Agency of the Czech Republic.

Development, validation and application to real samples of a multiresidue LC-MS/MS method for determination of β2 -agonists and anabolic steroids in bovine hair.

β(2) -agonists are often abused in cattle breeding because of their effects on animal growth and meat properties. The use of β(2) -agonists as growth promoters is forbidden in the European Union (Council Directive 96/23/EC classifies them into group A of Annex I), due to their toxicity and carcinogenic properties, as for anabolic steroids, which are often administered in combination with β(2) -agonists, to promote the storage of proteins and increase muscle size. A unique confirmatory liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative detection of 13 β(2) -agonists and anabolic steroids plus the qualitative identification of other three analytes in bovine hair was developed and validated, according to Decision 2002/657/CE. Hair samples were washed with dichloromethane, digested within a NaOH solution and subjected to liquid-liquid extraction. The analysis was performed by high performance liquid chromatography coupled to a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with good repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes. The quantitative calibrations obtained from spiked blank hair samples proved linear in the range tested. CCα and CCβ ranged from 0.5 ng/g to 30 ng/g. Intralaboratory reproducibility (CV%) ranged between 5.0 and 17.7 and trueness between 96% ± 7% and 105% ± 8%. The applicability of the method to real positive samples was demonstrated for both β(2) -agonists and anabolic steroids. 17α-boldenone was found in most (70%) hair samples obtained from untreated animals, supporting the hypothesis of endogenous production of this steroid.

Comparison of capillary electrophoresis–mass spectrometry and hydrophilic interaction chromatography–mass spectrometry for anionic metabolic profiling of urine

In order to assess the utility of a recently developed capillary electrophoresis–mass spectrometry (CE–MS) method for the study of anionic metabolites in urine, a comparison was made with hydrophilic interaction chromatography–MS (HILIC–MS) using negative electrospray ionization. After optimization of the HILIC conditions, a gradient employing 10mM ammonium acetate (pH 6.8) in acetonitrile–water (5min 90% acetonitrile followed by 90%–50% acetonitrile in 10min) was selected, providing baseline separation of five representative anionic test metabolites. Relative standard deviations (RSDs) for HILIC retention times and peak areas were below 0.2% and 7.7%, respectively, and detection limits were in the range 0.04–2.21μM. Metabolites in rat urine could also be analysed in a reproducible way with retention time and peak area RSDs below 0.6% and 13.6%, respectively. The CE–MS and HILIC–MS methods were compared in terms of reproducibility, sensitivity, selectivity and coverage of the anionic urinary metabolome. In general, peak area RSDs were similar whereas HILIC–MS yielded better retention-time repeatability and up to 80 times lower detection limits (expressed in injected concentration) for test metabolites as compared to CE–MS. Rat urine analysis by HILIC–MS provided detection of 1360 molecular features compared to 347 molecular features revealed with CE–MS. Of these, a number of 144 molecular features were found with both HILIC–MS and CE–MS, which showed on average 10 times higher peak areas in HILIC–MS. The HILIC retention and CE migration times of the common features were clearly not correlated. The HILIC and CE behavior of the test metabolites and 16 putatively identified common features were evaluated involving their physicochemical properties, indicating a markedly different separation selectivity, and thus significant degree of orthogonality of HILIC and CE.

Determination of furfurals in Manuka honey using piston-cylinder liquid–liquid extraction and gas chromatography

A rapid analytical approach for the direct measurement of furfurals such as 2-furfural and 5-methyl-2-furfural at parts-per-billion level in Manuka honey is described. The approach employs a piston-cylinder based liquid–liquid extraction device using chloroform extraction solvent. This device substantially reduces extraction time by a factor of 120 times compared to solid phase micro-extraction and reduces solvent consumption by a factor of 25 times compared to liquid–liquid extraction with mechanical agitation. A recently commercialised capillary column offering a high degree of inertness permits separation and detection of the analytes at ultra-trace level without derivatisation. A three-port planar microfluidic device with a mid-point pressure is also incorporated to back-flush heavier compounds in the matrix to improve column longevity and overall system cleanliness. With this approach, analysis is conducted in less than 7min. Repeatability of retention times for all compounds is less than 0.1% (n=20). The compounds cited can be analysed over a range from 1ng/g to 10μg/g in honey with a 5ng/g limit of quantification (LOQ) and correlation coefficients of at least 0.999. Relative precision is less than 2.8% RSD (n=20) at 50ng/g level with analyte extraction efficiency of greater than 99% (n=3) over a range from 5ng/g to 10μg/g in the matrix described. The analytical system requires only minimal maintenance and is suitable for remote site deployment. Under the analytical conditions established and with a practical LOQ of 5ng/g, 100 samples can be analysed before septum/liner/o-ring replacements are needed. As a preventive measure, the pre-column can be replaced once every six months to maintain chromatographic fidelity.

Eluent-Induced Separation of Inorganic Cations in Capillary Liquid Chromatography with Contactless Conductivity Detector

A non-suppressed contactless conductivity detector has been used as a capillary detector in a capillary ion chromatograph, combining a reversed-phase C30 column permanently modified with ionic surfactant. The C30 column (100 × 0.32 mm. id) was modified with sodium dodecyl sulfate (SDS) for the separation of inorganic cations. Monovalent cations could be separated by the proposed system, in which methanesulfonic acid (MSA) and SDS were employed as the mobile phase component, but divalent cations could not be eluted under this condition. As for the case of SDS used as the eluent, an H+-cation-exchange column was placed before the sample injector to convert the Na+ from the eluent into H+, and when the mixture of MSA and dodecyl sulfuric acid was used as the eluent, the retention of cations was improved and baseline separation of the cations was achieved within 23 min. The effect of the eluent composition on the retention behavior of inorganic cations was investigated. The repeatability of retention time and peak height varied from 0.39 to 0.58 and 2.21 to 3.25 % as relative standard deviation, respectively.

Evaluation of anti-inflammatory activity and fast UHPLC–DAD–IT-TOF profiling of polyphenolic compounds extracted from green lettuce (Lactuca sativa L.; var. Maravilla de Verano)

Fresh cut vegetables represent a widely consumed food worldwide. Among these, lettuce (Lactuca sativa L.) is one of the most popular on the market. The growing interest for this “healthy” food is related to the content of bioactive compounds, especially polyphenols, that show many beneficial effects. In this study, we report the anti-inflammatory and antioxidant potential of polyphenols extracted from lettuce (var. Maravilla de Verano), in J774A.1 macrophages stimulated with Escherichia coli lipopolysaccharide (LPS). Lettuce extract significantly decreased reactive oxygen species, nitric oxide release, inducible nitric oxide synthase and cycloxygenase-2 expression. A detailed quali/quantitative profiling of the polyphenolic content was carried out, obtaining fast separation (10min), good retention time and peak area repeatability, (RSD% 0.80 and 8.68, respectively) as well as linearity (R2⩾0.999) and mass accuracy (⩽5ppm). Our results show the importance in the diet of this cheap and popular food for his healthy properties.

Constant pressure mode extended simple gradient liquid chromatography system for micro and nanocolumns

Performing gradient liquid chromatography at constant pressure instead of constant flow rate has serious potential for shortening the analysis time and increasing the productivity of HPLC instruments that use gradient methods. However, in the constant pressure mode the decreasing column permeability during a long period of time negatively affects the repeatability of retention time. Thus a volume-based approach, in which the detector signal is plotted as a function of retention volume, must be taken into consideration. Traditional HPLC equipment, however, requires quite complex hardware and software modifications in order to work at constant pressure and in the volume-based mode. In this short communication, a low cost and easily feasible pressure-controlled extension of the previously described simple gradient liquid chromatography platform is proposed. A test mixture of four nitro esters was separated by 10–60% (v/v) acetone/water gradient and a high repeatability of retention volumes at 20MPa (RSD less than 0.45%) was realized. Separations were also performed at different values of pressure (20, 25, and 31MPa), and only small variations of the retention volumes (up to 0.8%) were observed. In this particular case, the gain in the analysis speed of 7% compared to the constant flow mode was realized at a constant pressure.

Suitability of postcolumn oxidation liquid chromatography method AOAC 2011.02 for monitoring paralytic shellfish toxins in Alaskan shellfish--initial pilot study versus mouse bioassay and in-house validation.

An in-house study was conducted to confirm the suitability of the postcolumn oxidation (PCOX) LC method, AOAC 2011.02, for regulatory monitoring of paralytic shellfish toxins (PSTs) in Alaskan shellfish. The following parameters were evaluated: calibration linearity, linear range, retention time stability, accuracy, repeatability, LOQ, and comparability with AOAC 959.08, the mouse bioassay (MBA) for PSTs. Mean recoveries for oyster homogenate spiked at 66 microg saxitoxin (STX) diHCI eq./100 g and 5 microg STX diHCI eq./100 g were 95 (n = 8) and 104%, respectively. Repeatability (n = 8) was 1.6 and 7% RSD, respectively. LOQ was estimated at 5 microg STX diHCl eq./100 g with S/N = 6 for STX. Comparability with the MBA was evaluated by duplicate analysis of regulatory samples using the original homogenate for both methods. Forty-one samples with MBA results varying between 40 and 500 microg STX diHCl eq./100 g were compared to corresponding PCOX values. The correlation coefficient (r2) = 0.96, with a slope of 2.1. The slope indicates an average 2X higher total toxicity result for PCOX versus MBA, a result that is consistent with prior literature showing low recoveries using the MBA.

HPCE AND HPLC METHODS FOR DETERMINATION OF CLOPIDOGREL AND ITS CARBOXYLIC ACID METABOLITE IN BIOLOGICAL SAMPLES: A COMPARATIVE ANALYSIS

A comparison between two analytical techniques (HPCE and HPLC) with UV detection for the determination of clopidogrel (CLP) and its carboxylic acid metabolite (CLPM) in biological fluids was performed. Resolution of the analytes by HPLC method was obtained on a C18 column with the use of a mobile phase consisted of acetonitrile and 0.03 M phosphate buffer at pH 3.0. HPCE analysis was performed in an uncoated capillary filled with an 0.025 M phosphate buffer at pH 2.5. A comparison of the both methods was performed in terms of validation parameters. Statistical analysis, Bland-Altman method, and ATE/LER zone method were also applied for assessing agreement between two methods. It was concluded that the HPLC method produced comparable results to the HPCE method with respect to the analysis of CLPM in plasma samples. Moreover, HPCE offered several advantages such as a lower solvent consumption and a higher selectivity of the analytes in plasma and urine samples. On the other hand, HPLC displayed excellent retention time repeatability in comparison to HPCE method. This study highlights the effective possibilities of application of both methods in the analysis of CLPM in plasma samples obtained from patients undergoing CLP therapy prior to percutaneous coronary intervention.

Ultra-fast separation of estrogen steroids using subcritical fluid chromatography on sub-2-micron particles

Estrogen steroids, represented by estradiol and its related substances, include both structurally very close and simultaneously different analogs. Their separation still remains an analytical challenge. Subcritical fluid chromatography (SbFC) on sub-2-micron particles was found to be an appropriate tool to obtain fast and efficient separation of nine target analytes. Among the four tested stationary phases charged hybrid modified with PFP (pentafluorophenyl) moiety was found to be the most convenient providing the fastest separation within 1.6 min using quick gradient elution with carbon dioxide and methanol as an organic modifier. However, complete separation was obtained also on other tested phases including bare hybrid stationary phase, hybrid stationary phase modified with 2-EP (2-ethylpyridine) and also C18, which is less typical in SbFC. The baseline separation on the latter columns was achieved by means of a temperature increase, a change in organic modifier type and gradient time increase respectively. Quantitative performance was evaluated at optimized conditions and method validation was accomplished. Excellent repeatability of both retention times (RSD<0.15%) and peak areas (RSD<1%) was observed. The method was linear in the range of 1.0-1000.0 μg/ml for all steroids with the lowest calibration point being an LOQ, except for Δ-derivatives, that provided better sensitivity and thus LOQ of 0.5 μg/ml. The sensitivity was sufficient for the analysis of real samples although it was still five times lower compared to UHPLC-UV experiments.