Performance Liquid Chromatography Research Articles

Evaluation of the Triglyceride Composition of Pomegranate Seed Oil by RP‐HPLC Followed by GC‐MS

AbstractTriglyceride composition and fatty acid profiles of pomegranate seed oil were evaluated by newly developed methods in reverse‐phase‐high performance liquid chromatography (RP‐HPLC) and gas chromatography (GC), respectively. Different compositions of the mobile phase (acetone and acetonitrile) and flow rates for the HPLC system were used to obtain better separation for accurate quantitative analysis. Triglycerides with conjugated fatty acids (CLnAs) were eluted in order of the polarity of their geometrical isomers (c, t, c < t, t, c < t, t, t). The dominant triglyceride was found to be PuPuPu (32.99 %) in pomegranate seed oil, followed by PuPuCa and PuCaCa containing punicic acid and catalpic acid with total triglyceridelevels of 27.72 and 10.11 %, respectively. For fatty acid composition analysis, triglyceride fractions were derivatized into their respective methylesters which were injected into gas chromatography‐mass spectrometry (GC‐MS) to identify and gas chromatography‐flame ionization detector (GC‐FID) to quantify the conjugated fatty acids of each fraction of triglycerides. Punicic acid was found to be dominant (76.57 %) followed by catalpic acid (6.47 %) and β‐eleotearic acid (1.45 %). Pomegranate seed contained greater amounts of conjugated linolenic acids. These results showed that the present study provides more information about the composition of the triglyceride and fatty acid profiles of pomegranate seed oil compared to the reported studies. Therefore, the developed methods in this study can be used for the identification of the triglyceride and fatty acid composition for pomegranate seed oils and some such specials edible oils including CLnA isomers.

Saxitoxin and the Ochre Sea Star: Molecule of Keystone Significance and a Classic Keystone Species.

Saxitoxins (STXs) are paralytic alkaloids produced by marine dinoflagellates in response to biotic and abiotic stressors yielding harmful algal blooms. Because STX impacts coastal, near-shore communities to a greater extent than would be predicted by its relative abundance, it has been referred to as a "molecule of keystone significance" in reference to Robert Paine's Keystone Species Concept. Pisaster ochraceus, the predator upon which Paine's concept was founded, inhabits waters regularly plagued by harmful algal blooms, but the effects of STX on Pisaster have not yet been investigated. Here, we used laboratory and field experiments to examine the potential consequences of exposure to STX on sea stars' feeding, attachment to the substrate, and success in fertilization. Pisaster exhibited similar feeding behaviors when offered non-toxic prey, STX-containing prey, or a combination of the two. Although feeding behavior is unaffected, consumption of STX poses a physiological tradeoff. Sea stars in the laboratory and field had significantly lower thresholds of the force needed to detach them from their substrates after either being exposed to, or consuming, STX. High pressure (or high performance) liquid chromatography analysis indicated an accumulation of STX (and structural analogues) in sea stars' viscera, likely due to trophic transfer from toxic prey. Incidence of fertilization tended to decrease when gametes were exposed to high, yet ecologically relevant, STX concentrations of STX. These findings suggest that the molecule of keystone significance, STX, produced during harmful algal blooms extends its impacts to rocky intertidal communities by way of the keystone predator P. ochraceus.

Characterization of Bioactive compound isolated from Myrothecium spp. with UV, FTIR and HPLC Analysis

Development of new drugs, especially in area of infectious diseases, represents today one of the most important research. Fungal isolates are receiving increasing attention by natural product chemists due to their diverse and structurally unprecedented compounds making them interesting candidates for drug discovery. In fact, need for novel, safe and more efficient antibiotics is a key challenge to the pharmaceutical industry today, moreover, increase in opportunistic infections in the immune compromised host has influenced this demand. Nowadays, evaluating morphological and biochemical differences as well as studying fungal genetic diversity via molecular indicators seem to be the most common method for screening this genus. In this research we evaluate the potential of bioactive compound, production and characterize the UV and FTIR spectroscopy and HPLC (High performance liquid chromatography) analysis pattern of isolated Myrothecium spp. MRP001. Process development for high level production of bioactive compound was applied using OFAT method. Then, following the extraction of secondary metabolite, the UV and FTIR spectroscopy analysis was carried out for characterization of the various extracts. Considering the coordinate analysis of UV and FTIR spectroscopy pattern, the isolate MRP001 with substantial antimicrobial activity exhibited absorption at 3411 cm-1 which is indicator of hydroxyl groups, absorption at 2856 and 2915 cm-1 indicating hydrocarbon chassis, and absorption at 1649 cm-1 indicating a double bond of polygenic compound. These results highlight the importance of Myrothecium isolates in antibiotic production. HPLC confirmed the production when compared with standards

Efficacy of albendazole chitosan microsphere against echinococcus multilocularis infection in mice

Objective To observe the therapeutic efficacy of albendazole chitosan microsphere (ABZ- CS- MPs)in mice infected with echinococcus multilocularis(E.m). Methods To establish secondary intraperitoneal infections of E.m in mice model, the 80 mice infected echinococcus multilocularis for 12 weeks were randomly divided into four groups: ABZ- CS- MPs group, albendazole oral emulsion(ABZ- E)group,albendazole tablet(ABZ- T)group and model control group,above 3 experimental groups were oral administrated with 75 mg/kg dosage; model control group were administrated with 0.2 ml saline per mouse dosage; All groups were administered with 3 times a week medication for 10 weeks. Following investigating markers were monitored after overall administration:(1)observation of morphological changes;(2) Calculation of mean cyst weight and inhibition rate after treatment;(3)observation of Histopathological changes of the E.m vesicles; 4)Measurement of the concentrations of albendazole sulfoxide(ABZ- SX)in plasma and liver homogenates by high- performance liquid chromatography(HPLC). Results There were obvious collapse and calcification of E.m vesicles in ABZ- CS- MPs group, mean cyst weights of E.m tissues in each groups treated with ABZ were[(0.114±0.030),(0.247±0.086),(0.582±0.145)g], there were significant differences of mean cyst weights between ABZ treated groups and model control group(P< 0.05), ABZ- CS- MPs had a higher suppressive effect on vesicular development than those ABZE treated group and ABZ- T treated group, inhibition rate of E.m vesicles in those 3 groups were: 91.04%, 80.60%, 54.28% respectively. Hematoxylin and eosin(HE) staining showed that there was different level of degeneration, necrosis on experimental groups, compared with other groups, germinal layer and laminate layer have severest damages in ABZ- CS- MPs treated group.Concentration of ABZ- SX in plasma and liver are much higher than ABZ- T group(P<0.01). Conclusion It is concluded that ABZ- CS- MPs had higher inhibitory effect against mice infected with alveolar echinococcosis, it promoted the intestinal absorption of ABZ effectively and further increased the drug concentration in plasma and liver. Key words: Alveolar echinococcosis; Chitosan microsphere; Albendazole; Mice

The hand eczema proteome: imbalance of epidermal barrier proteins.

Chronic hand eczema (CHE) is a common skin disease with a high socioeconomic impact. While some light has been shed on the genetic factors that predispose individuals to the disease, little is known about its actual pathogenesis. We aimed to carry out a systematic and comprehensive analysis of the differential protein expression in CHE using modern mass spectrometry. We performed liquid chromatography with tandem mass spectrometry analyses and label-free quantification to analyse the proteomic profile of palmar skin from 12 individuals (six patients with hand eczema and six healthy volunteers). Immunohistochemistry of the palmar skin from seven different patients with hand eczema and seven different healthy volunteers was performed in a second step. With this method we were able to identify 185 candidate proteins with a significantly different abundance in the hand eczema samples. Among them we found several barrier proteins: filaggrin (FLG), FLG-2 and hornerin were all downregulated in the hand eczema samples, as were the desquamation-related enzymes kallikrein-related peptidase (KLK)5 and KLK7 and cystatin E/M. The antimicrobial peptides S100A7 and S100A8/A9 and the small proline-rich protein 2B and S100A11 were upregulated in the diseased skin. Immunohistochemistry confirmed these findings. Our results corroborate the assumption that skin barrier dysfunction plays an essential role in the pathogenesis of CHE.

Optimization of Data Acquisition and Sample Preparation Methods for LC-MS Urine Metabolomic Analysis

Abstract Nowadays, chromatographic methods coupled with mass spectrometry are the most commonly used tools in metabolomics studies. These methods are currently being developed and various techniques and strategies are proposed for the profiling analysis of biological samples. However, the most important thing used to maximize the number of entities in the recorded profiles is the optimization of sample preparation procedure and the data acquisition method. Therefore, ultra high performance liquid chromatography coupled with accurate quadrupoletime- of-flight (Q-TOF) mass spectrometry was used for the comparison of urine metabolomic profiles obtained by the use of various spectral data acquisition methods. The most often used method of registration of metabolomics data acquisition – TOF (MS) was compared with the fast polarity switching MS and auto MS/MS methods with the use of multivariate chemometric analysis (PCA). In all the cases both ionization mode (positive and negative) were studied and the number of the identified compounds was compared. Additionally, various urine sample preparation procedures were tested and it was found that the addition of organic solvents to the sample noticeably reduces the number of entities in the registered profiles. It was also noticed that the auto MS/MS method is the least efficient way to register metabolomic profiles.

The effects of micelles of differently charged surfactants on the equilibrium between (Z)- and (E)-diastereomers of five ACE inhibitors in aqueous media

The (Z)/(E) equilibria in water solutions of five ACE inhibitors (captopril, enalapril, lisinopril, perindopril, and ramipril) in presence and in absence of surfactants (anionic sodium dodecyl sulfate, cationic cetyltrimethyl ammonium bromide, and non-ionic 4-octylphenol poly- ethoxylate) were analyzed by reversed-phase high- performance liquid chromatography. The results showed that the (Z)/(E) equilibrium of lisinopril and captopril was the least sensitive to the effect of the examined surfactants. 4-Octylphenol polyethoxylate expressed the weakest effect on isomerization of the ACE inhibitors, while the (Z)/ (E) equilibria of enalapril, ramipril, and perindopril were the most sensitive to the presence of sodium dodecyl sul- fate and cetyltrimethyl ammonium bromide. In addition, the response of two structurally very close compounds, such as enalapril and ramipril to the presence of sodium dodecyl sulfate and cetyltrimethyl ammonium bromide was opposite, and these two ACE inhibitors had different chromatographic behavior. To provide a better insight into the reasons for the differences in order of elution of (Z)- and (E)-diastereomers and the possible molecular mecha- nism underlying their retention, (Z)- and (E)-diastereomers of enalapril and ramipril were subjected to a theoretical study optimized at the B3LYP/6-31G (d,p) level of den- sity functional theory. The Connolly solvent-excluded volume was taken as the most significant parameter that can be connected to differences in chromatographic behavior of the (Z)- and (E)-diastereomers of enalapril and ramipril. Graphical abstract

BisGMA and TEGDMA Elution from Two Flowable Nanohybrid Resin Composites: An In vitro Study

Aim: The aim of our study was to evaluate the amount of release of BisGMA and TEGDMA from two commercially available enamel replacement composites; Tetric N-FlowTM and G-aenial Universal FloTM over a period of 24 hours after polymerization with a standard LED Curing Unit. Methods and Materials: Two flowable nanohybrid composite materials; Tetric N-FlowTM (Ivoclar Vivadent AG, Liechtenstein) and G-aenial Universal FloTM (GC, India) were investigated and grouped into two groups. Ten samples from each group were prepared by inserting the material into a standardized Teflon mould of size 2x2x2 mm. Each sample was cured with a LED curing unit for 20 seconds and was stored in 2 ml of Ethanol at room temperature. After 24 hours, the samples were removed from the storage medium (ethanol) and prepared for measurements. A reverse phase HPLC unit was used to detect the release of BisGMA and TEGDMA monomers from the prepared samples. The acquired measurements were obtained after testing them in a High Liquid Performance Chromatography Unit. The data obtained was statistically analyzed and the results revealed significant amount of release of TEGDMA as well as BisGMA. Original Research Article Hegde and Wali; BJMMR, 5(9): 1096-1104, 2015; Article no.BJMMR.2015.123 1097 Results: G-aenial Universal FloTM showed significant release of both TEGDMA as well as BisGMA as compared to Tetric NFlowTM. The increase was by 0.5 units. Conclusion: Significant amount of release of TEGDMA as well as BisGMA was seen in both the composite materials after HPLC Unit analysis. This can help in the evaluation of cytoxicity to the soft tissues in the oral environment.

Ultrahigh-performance liquid chromatography-ion trap mass spectrometry characterization of the steroidal saponins of Dioscorea panthaica Prain et Burkill and its application for accelerating the isolation and structural elucidation of steroidal saponins

Dioscorea panthaica is a traditional Chinese medicinal herb used in the treatment of various physiological conditions, including cardiovascular disease, gastropathy and hypertension. Steroidal saponins (SS) are the main active ingredients of this herb and have effects on myocardial ischemia and cancer. The phytochemical evaluation of SS is both time-consuming and laborious, and the isolation and structural determination steps can be especially demanding. For this reason, the development of new methods to accelerate the processes involved in the identification, isolation and structural elucidation of SS is highly desirable. In this study, a new ultrahigh performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT/MSn) method has been developed for the identification of the SS in D. panthaica Prain et Burkill. Notably, the current method can distinguish between spirostanol and furostanol-type compounds based on the fragmentation patterns observed by electrospray ionization-ion trap mass spectrometry (ESI-IT/MSn) analysis. UHPLC-IT/MSn was used to conduct a detailed investigation of the number, structural class and order of the sugar moieties in the sugar chains of the SS present in D. panthaica. The established fragmentation features were used to analyze the compounds found in the 65% ethanol fraction of the water extracts of D. panthaica. Twenty-three SS were identified, including 11 potential new compounds and six groups of isomers. Two of these newly identified SS were selected as representative examples, and their chemical structures were confirmed by 1H and 13C NMR analyses. This newly developed UHPLC-IT/MSn method therefore allowed for the efficient identification, isolation and structural determination of the SS in D. panthaica.

Antihypertensive peptides from curd.

Introduction:Curd (Dadhi) peptides reduce hypertension by inhibiting angiotensin converting enzyme (ACE) and serum cholesterol. Peptides vary with bacterial species and milk type used during fermentation.Aim:To isolate and assay the antihypertensive peptides, before and after digestion, in two commercially available curd brands in Sri Lanka.Materials and Methods:Whey (Dadhi Mastu) separated by high-speed centrifugation was isolated using reverse-phase-high- performance liquid chromatography (HPLC). Eluted fractions were analyzed for ACE inhibitory activity using modified Cushman and Cheung method. Curd samples were subjected to enzymatic digestion with pepsin, trypsin, and carboxypeptidase-A at their optimum pH and temperature. Peptides isolated using reverse-phase-HPLC was assayed for ACE inhibitory activity.Results:Whey peptides of both brands gave similar patterns (seven major and five minor peaks) in HPLC elution profile. Smaller peptides concentration was higher in brand 1 and penta-octapeptides in brand 2. Pentapeptide had the highest ACE inhibitory activity (brand 2–90% and brand 1–73%). After digestion, di and tri peptides with similar inhibitory patterns were obtained in both which were higher than before digestion. Thirteen fractions were obtained, where nine fractions showed more than 70% inhibition in both brands with 96% ACE inhibition for a di-peptide.Conclusion:Curd has ACE inhibitory peptides and activity increases after digestion.

In Vitro Antimicrobial Efficacy of Fractions from Onion (Allium Cepa) Leaves Extract from Wukro, Ethiopia

To examine the in vitro anti bacterial activities of the ethanol extract of fresh leaves of Alliumcepa (onion) and to determine and quantify the phenol compounds of the investigated plantparts. This study was drifting out at the Mekelle university department chemistry and Adigrat pharmaceutical industry, fromMarch2015toApril 2015. Clinical strains of Streptococcus pneumoniae and ethanol extracts of the plant species was used for the antimicrobial study. Thirty grams of the sample was ground, filtrated, and each filtrate mixed with 100 ml ethanol and placed in a shaker for 48 hours. Theethanol was evaporated from the sample, weighed, and subjected to an antibacterial activity test using the agar diffusion method. The high- performance liquid chromatography was used to identify and quantify the phenols extracts of investigated samples. Ethanol extract of the investigated plant parts showed antibacterial activities against different pathogenicbacteria. Leaf extracts of Alliumcepashowed the highest antibacterial activity and contains more phenols. Theethanol extract of the tested plants could be considered as analternative source of new antibacterialdrugs.

Pharmacokinetics of Chloroquine and Metronidazole in Rats

Article history: The single oral dose pharmacokinetics of chloroquine (5mg/kg body weight) and metronidazole (7.5mg/kg body weight) were studied in rats' serum. Chloroquine and metronidazole concentrations were measured using high - performance liquid chromatography (HPLC) method developed earlier in our laboratory. The data were fitted into a WinNonlin standard non-compartmental programme. The Maximum serum concentration Cmax (µg/ml) of chloroquine was 5.70 ± 1.41 while that of metronidazole was 3.13  0.30, Time to peak concentration tmax was 1.00  0.00 (h) and that of metronidazole was 0.83 ± 0.27, Volume of distribution Vd (L) 1.33 ± 0.26 for metronidazole 2.39  0.28; Elimination half-life t1/2 (h) 10.05 ± 3.01 for metronidazole 4.05  0.46. The values were comparable with the works of other authors. Compounds that show very high activity in -vitro may not have in vivo activity, or may be highly toxic using in- vivo models due to undesirable pharmacokinetic properties, and toxicity may result from formation of reactive metabolites. This study assures the quality of the brands of the drugs and encouraged the use of animal model in determining pharmacokinetic properties especially in drug design.

Water Uptake and Urea Release of Hydrogels Based on Acrylamide, Methyl Methacrylate and N,N’–Methylene–bis–Acrylamide

Water swelling kinetics, equilibrium water content, urea release kinetics, and freezable and non–freezable water of hydrogels based on acrylamide (AM), methyl methacrylate (MMA) and N,N’–methylene–bis– acrylamide (NMBA) were studied. A series of copolymeric gels of AM, MMA and NMBA as a crosslinker agent were prepared by free radical solution copolymerization using a mixture of ethanol and water as a solvent and benzoyl peroxide (BPO) as initiator. Equilibrium water content (Wω) was strongly influenced by copolymer composition. For hydrogels richer in AM, EWC increased as crosslinking decreased. Instead, for high content methacrylate hydrogels, the hydrophilicity of NMBA decreased Wω. Swelling process in distilled water was studied by dynamic swelling measurements (gravimetric method ). It was found that synthesized hydrogels fits second–order swelling. Urea release studies were carried out in distilled water and the urea concentration was determined by High Performance Liquid Chroma togra phy (HPLC). The hydrogels del ivered urea very ra pidl y, for all the studied compositions most of the urea was released in less than 200 minutes. Differential Scanning Calorimetry (DCS) was employed to determine the relative proportions of non–freezing and freezing water. DSC traces shown multiple endotherms that were related with different states of association between water and the polymeric matrix.

Metabolite profiling in posttraumatic stress disorder.

BackgroundTraumatic stress does not only increase the risk for posttraumatic stress disorder (PTSD), but is also associated with adverse secondary physical health outcomes. Despite increasing efforts, we only begin to understand the underlying biomolecular processes. The hypothesis-free assessment of a wide range of metabolites (termed metabolite profiling) might contribute to the discovery of biological pathways underlying PTSD.MethodsHere, we present the results of the first metabolite profiling study in PTSD, which investigated peripheral blood serum samples of 20 PTSD patients and 18 controls. We performed liquid chromatography (LC) coupled to Quadrupole/Time-Of-Flight (QTOF) mass spectrometry. Two complementary statistical approaches were used to identify metabolites associated with PTSD status including univariate analyses and Partial Least Squares Discriminant Analysis (PLS-DA).ResultsThirteen metabolites displayed significant changes in PTSD, including four glycerophospholipids, and one metabolite involved in endocannabinoid signaling. A biomarker panel of 19 metabolites classifies PTSD with 85% accuracy, while classification accuracy from the glycerophospholipid with the highest differentiating ability already reached 82%.ConclusionsThis study illustrates the feasibility and utility of metabolite profiling for PTSD and suggests lipid-derived and endocannabinoid signaling as potential biological pathways involved in trauma-associated pathophysiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s40303-015-0007-3) contains supplementary material, which is available to authorized users.

SUPERCRITICAL FLUIDS AND ULTRASOUND ASSISTED EXTRACTIONS APPLIED TO SPRUCE BARK CONVERSION

Supercritical fluid and ultrasound assisted extractions were applied as a first step in a complex processing of spruce bark in order to recover polyphenols compounds. The aim of the study was to compare the efficiency of these green processes with a conventional extraction technique (ethanolic extraction). Supercritical fluid extraction was carried out in two steps: (i) static extraction for 15 min at 1000 psi with pure CO2 and (ii) dynamic extraction for 45 min at 35, 40 and 50 0 C, 1200, 2000 and 2500 psi, with CO2 and 70% ethanol as co-solvent. UAE was carried out in an ultrasonic bath at 45, 50 and 60 0 C for 5 to 75 minutes. The ethanolic extraction was performed using ethanol (70%) in a closed oven for 13 days. The extracts were characterized using Folin-Ciocalteu method for total phenolic content and quantified by high liquid performance chromatography (HPLC). The study recommend SFE and UAE instead of traditional ethanolic techniques since these provide high extraction yields, pure extracts, with a large number of polyphenolic compounds extracted and are environmentally friendly.

Fast screening of 24 sedative hypnotics illegally added in improving sleep health foods by high performance liquid chromatography-ion trap mass spectrometry

A fast screening method was established for the simultaneous determination of 24 sedative hypnotics illegally added in improving sleep health foods by high performance liquid chromatography-ion trap mass spectrometry (HPLC-IT MS). The method was based on the sonication assisted extraction of the improving sleep health food samples using methanol. The extract was then filtrated with 0.45 µm filter membrane and the filtrate was separated on a Phenomenex Luna C18 column with isocratic elution at a flow rate of 0.3 mL/min. A binary mobile phase was 0.05% (v/v) formic acid (solvent A)-methanol/acetonitrile (15:25, v/v, solvent B). The electrospray ionization (ESI) source in positive ion mode or negative ion mode was used to scan MS1-MS3 spectra for the 24 sedative hypnotics. The MS2 and MS3 spectra were used for qualitative analysis of samples. The calibration graphs were linear in their concentration ranges with the correlation coefficients (r2) more than 0.999. The limits of detection (LODs) were 4.0-446.6 µg/L. The recoveries for all the drugs in the improving sleep health foods were 88.6%-110.3% with the relative standard deviations no more than 9.8% at three spiked levels. Twenty-seven batches of the improving sleep health foods were tested. Melatonin was found in eighteen batches. The method is fast, specific, sensitive, easy and suitable for fast screening of 24 sedative hypnotics illegally added in improving sleep health foods.

Liquid chromatography and liquid chromatography-mass spectrometry analysis of donepezil degradation products

This study describes the investigation of degradation products of donepezil (DP) using stability indicating RP-HPLC method for determination of donepezil, which is a centrally acting reversible acetylcholinesterase inhibitor. In order to investigate the stability of drug and formed degradation products, a forced degradation study of drug sample and finished product under different forced degradation conditions has been conducted. Donepezil hydrochloride and donepezil tablets were subjected to stress degradation conditions recommended by International Conference on Harmonization (ICH). Donepezil hydrochloride solutions were subjected to acid and alkali hydrolysis, chemical oxidation and thermal degradation. Significant degradation was observed under alkali hydrolysis and oxidative degradation conditions. Additional degradation products were observed under the conditions of oxidative degradation. The degradation products observed during forced degradation studies were monitored using the high performance liquid chromatography (HPLC) method developed. The parent method was modified in order to obtain LC-MS compatible method which was used to identify the degradation products from forced degradation samples using high resolution mass spectrometry. The mass spectrum provided the precise mass from which derived molecular formula of drug substance and degradation products formed and proved the specificity of the method unambiguously.

Poor performance of laboratories assaying newly developed antiretroviral agents: results for darunavir, etravirine, and raltegravir from the international quality control program for therapeutic drug monitoring of antiretroviral drugs in human plasma/serum.

The International Interlaboratory Quality Control PROGRAM for Therapeutic Drug Monitoring of Antiretroviral Drugs in Human Plasma/Serum was initiated in 1999. We have previously published our experience during the first 10 years of the PROGRAM. Since 2010, 3 newly developed antiretroviral agents have been added to the darunavir, etravirine, and raltegravir. The objective of this analysis is to describe the performance of participating laboratories measuring these newer agents in 2011-2012. Each year, laboratories received 2 blind samples of human plasma/serum spiked with a low (<1.0 mg/L), medium (1.0-5.0 mg/L), or high (>5.0 mg/L) concentration of these drugs. Laboratory results were standardized to percentages with reference to the nominal (true) concentration. Any result that deviated more than 20% of the nominal values was defined as inaccurate. The numbers of laboratories that participated by the end of 2012 were 44 for darunavir, 28 for etravirine, and 30 for raltegravir. A total of 357 results were evaluable for analysis. Of these, 64 (17.9%) results were reported with >20% deviation, so "inaccurate" (7.6% too low, 10.4% too high). The proportion of inaccurate results in 2011 was 21.3% for darunavir, 31.0% for etravirine, and 26.3% for raltegravir; in 2012, these figures improved to 8.1%, 23.2%, and 8.3% for darunavir, etravirine, and raltegravir, respectively. Taking darunavir as the reference, performance for etravirine was significantly lower [odds ratio = 0.462, 95% confidence interval: 0.246-0.866, P = 0.016] and performance for raltegravir was not significantly different. Low concentrations were significantly more frequently reported as inaccurate than medium or high concentrations: 28.6% versus 10.6% versus 8.8%, respectively (P < 0.001). Laboratories that used Liquid Chromatography with tandem Mass Spectrometry did not perform better than those using High Performance Liquid Chromatography/Ultrarapid Performance Liquid Chromatography: 41 inaccurate results in 200 samples (20.5%) versus 23 in 157 samples (14.6%, P = 0.154). Multiple logistic regression revealed that the concentration range was the only significant predictor of inaccurate results. The lower range of concentrations performed worse than medium or high concentrations (P < 0.001). Laboratories continue to have problems with adequately measuring low plasma concentrations of antiretroviral agents. This is particularly a problem for some of the newer antiretroviral agents with plasma concentrations in the <1.0 mg/L range, such as etravirine and raltegravir.

Profiling of grape monoterpene glycosides (aroma precursors) by ultra-high performance-liquid chromatography-high resolution mass spectrometry (UHPLC/QTOF).

A 'suspect screening analysis' method for grape metabolomics by ultra-high performance-liquid chromatography (UHPLC) and high-resolution quadrupole-time of flight (QTOF) mass spectrometry was recently developed. This method was applied to study grape monoterpene glycosides, the main grape aroma precursors. Since standard compounds were not available, they were tentatively identified by overlapping various analytical approaches, in agreement with the indications recommended in mass spectrometry (MS)-based metabolomics. Accurate mass and isotopic pattern, MS/MS fragmentation, correlation between fragments observed and putative structures and between liquid chromatography coupled with mass spectrometry (LC/MS) and gas chromatography/mass spectrometry signals were studied. Seventeen monoterpene glycosides were identified without performing the hydrolytic artifacts commonly used to study these compounds which may affect sample profile. This is the first time that a detailed study of these aroma precursors has been carried out by direct LC/MS analysis.

Close relationship between redox state of human serum albumin and serum cysteine levels in non-diabetic CKD patients with various degrees of renal function.

Human mercaptalbumin (HMA) acts as a covalent-carrier protein for sulfur-containing amino acids, cysteine, and glutathione. In addition, its sulfhydryl residue reacts with peroxyl radicals. In this study, we evaluated the redox state of human serum albumin and its relationship with serum amino thiol levels in non-diabetic chronic kidney disease (CKD) patients with various degrees of renal function. High performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring were used to analyze serum samples collected from 37 non-dialysis patients with primary glomerulonephritis without diabetes mellitus (DM) or other systemic diseases (non-diabetic CKD patients). Total cysteine and homocysteine plasma levels increased with decreasing renal function and showed a significant negative correlation with glomerular filtration rate. The protein-bound ratio of serum cysteine also changed with the degree of renal dysfunction. The HPLC fraction of human mercaptalbumin (HMA) (%) was significantly lower in nondiabetic CKD patients than in healthy subjects. The redox state of human serum albumin (i.e., HMA %) correlated significantly with the total serum cysteine level. The HPLC fraction of HMA (%) closely correlated with the serum cysteine level in non-diabetic CKD patients. An increase in oxidized cysteine with impaired renal function and a reduced plasma redox capacity associated with a decrease in the reduced form of serum albumin (HMA %) may be important risk factors for promoting long-term complications in patients with renal dysfunction.