High-performance Liquid Chromatography Fractions Research Articles

Directly Coupled High-Performance Liquid Chromatography–Accelerator Mass Spectrometry Measurement of Chemically Modified Protein and Peptides

Quantitation of low-abundance protein modifications involves significant analytical challenges, especially in biologically important applications, such as studying the role of post-translational modifications in biology and measurement of the effects of reactive drug metabolites. (14)C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time (14)C quantitation of high-performance liquid chromatography (HPLC) separations by liquid sample accelerator mass spectrometry (LS-AMS). By enabling direct HPLC-AMS coupling, LS-AMS overcomes several major limitations of conventional HPLC-AMS, where individual HPLC fractions must be collected and converted to graphite before measurement. To demonstrate LS-AMS and compare the new technology to traditional solid sample AMS (SS-AMS), reduced and native bovine serum albumin (BSA) was modified by (14)C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 10(6) to 10(8) proteins. (14)C incorporated into modified BSA was measured by solid carbon AMS and LS-AMS. BSA peptides were generated by tryptic digestion. Analysis of HPLC-separated peptides was performed in parallel by LS-AMS, fraction collection combined with SS-AMS, and (for peptide identification) electrospray ionization and tandem mass spectrometry (ESI-MS/MS). LS-AMS enabled (14)C quantitation from ng sample sizes and was 100 times more sensitive to (14)C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol (14)C/peak. Additionally, LS-AMS turnaround times were minutes instead of days, and HPLC trace analyses required 1/6th the AMS instrument time required for analysis of graphite fractions by SS-AMS.

Bioactive Metabolites from Endophytes Associated with Rubiaceous Plants from Uitm Puncak Alam Biological Reserve

Plant endophytes are an important and novel resource of natural bioactive compounds. In the past two decades, many valuable bioactive compounds with antimicrobial, cytotoxic and anticancer activities have been successfully discovered from the endophytic microorganisms. In this research, 54 endophytic fungi and 6 endophytic bacteria were isolated from leaves, stems, flowers and roots of the three plants species of Rubiaceae family collected in the biological reserve, UiTM Puncak Alam, Malaysia. The objective of this study is to isolate bioactive metabolites of endophytic microorganisms from Rubiaceae plants. The microorganisms were inoculated and fermented according to a standardized procedure. Each culture was extracted using ethyl acetate based on our standard operation procedure. The crude extracts were evaluated for preliminary screening of antimicrobial activity against Staphylococcus aureus ATCC 25923, Enterococcus Faecium ATCC 51585, Pseudomonas Aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Candida Albicans ATCC 44831, using the MTT method. Pleasingly, the antimicrobial effects of these crude extracts were slightly lower than standard antibiotics. The minimum inhibitory concentration (MIC) of the crude ethyl acetate extracts ranged from 0.1875 mg/mL to 1.5 mg/mL. The crude extracts were analyzed by high performance liquid chromatography (HPLC). HPLC fractionation followed by retesting of the fraction led to correlate chromatographic peaks with biological activity. Pure compound was subjected to standard spectrometry analyzes and its structure established. Data of compounds will be presented.

Identification of compounds from the plant species Alepidea amatymbica active against HIV

As Alepidea amatymbica is commonly used and accepted as medicinal plant in South Africa for various indications, the scientific basis of its anecdotally described, putative anti-HIV properties was investigated. To this aim, we used an accelerated extraction–purification approach; extracts and therein sub-fractions of A. amatymbica were assessed in a cell-based assay targeting the replication of prototypic CXCR4-tropic (NL4-3) or CCR5-tropic (NL-AD87) HIV-1 strains.Sub-fractions of the extracts were generated through semi-preparative high performance liquid chromatography (HPLC) fractionation into triplicates of 96-well microtitre plates; they were then separately subjected to biological analysis and ultra performance liquid chromatography (UPLC) time-of-flight (TOF) analysis. A correlation plot was generated between the biological and chemical data to identify the biologically active compounds in those fractions that showed significant selective anti-HIV activity. The results indicated that rosmarinic acid was present in the wells that showed promising anti-HIV activity in vitro indicating that this compound is at least in part responsible for the antiviral properties of the A. amatymbica extracts. However, compared to standard retroviral inhibitor the anti-HIV activity of the pure compound was found to be only quite moderate. Nevertheless, the accelerated approach described herein increases the efficiency of screens towards identifying drug candidates much earlier in the discovery stage.

HPLC–MTT assay: Anticancer activity of aqueous garlic extract is from allicin

A strategy using reversed-phase high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrometry (MS), nuclear magnetic resonance (NMR), chemical synthesis, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay to identify allicin as the active anticancer compound in aqueous garlic extract (AGE) is described. Changing the pH of AGE from 7.0 to 5.0 eliminated interfering molecules and enabled a clean HPLC separation of the constituents in AGE. MTT assay of the HPLC fractions identified an active fraction. Further analysis by TLC, MS, and NMR verified the active HPLC fraction as allicin. Chemically synthesized allicin was used to provide further confirmation. The results clearly identify the active compound in AGE as allicin.

GC/MS analysis of high-performance liquid chromatography fractions fromSophora flavescensandTorilis japonicaextracts and theirin vitroanti-neosporal effects onNeospora caninum

We analyzed alcoholic extracts of herbs possessing anti-neosporal activity against Neospora (N.) caninum. To identify the chemical components of Sophora (S.) flavescens and Torilis (T.) japonica associated with anti-neosporal activity, specific fractions were isolated by high-performance liquid chromatography (HPLC). In vitro activity of the fractions against N. caninum was then assessed. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify specific anti-neosporal molecules in the herbal extracts. Almost all HPLC fractions of S. flavescens and T. japonica had higher levels of anti-neosporal activity compared to the not treated control. Active constituents of the extracts were sophoridane, furosardonin A, and tetraisopropylidene-cyclobutane in S. flavescens; 5,17-β-dihydroxy-de-A-estra-5,7,9,14-tetraene, furanodiene, and 9,12-octadecadienoic acid (Z,Z)-(CAS,1) in T. japonica.

Analysis of the Nitrogen Content of Distillate Cut Gas Oils and Treated Heavy Gas Oils Using Normal Phase HPLC, Fraction Collection and Petroleomic FT-ICR MS Data

The determination of the nitrogen content of petroleum products is important because nitrogen compounds decrease product quality and can be difficult to remove through processes such as hydrotreating. In recent years, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used to study the nitrogen species present in petroleum samples (petroleomics), with the goal of identifying hydrotreatment resistant species. While open column separations of petroleum samples are common, there has been little work done that employs a high-performance liquid chromatography (HPLC) separation prior to FT-ICR MS analysis. In this work, distillate cut and treated gas oils are separated on a commercially available dinitrophenyl “DNAP” column and the fractions are collected offline and analyzed by FT-ICR MS. HPLC separations on the “DNAP” column are shown, along with separations on a custom synthesized HPLC column (HC-Tol). Four peak regions are identified on the “DNAP” chromatograms, and the nitrogen species identified in each fraction using positive and negative electrospray ionization are discussed. It was found that pyridines are present in the first three fractions, while pyrroles are present in fractions 2 and 3. Acids are isolated in fraction 4. Alkylation of nitrogen species decreases their level of retention on the “DNAP” column, and the HPLC chromatograms can be used to qualitatively compare nitrogen content between samples. A comparison of HPLC fraction data to MS analysis of unfractionated gas oils found that fraction data are not adequate for quantitative comparisons of carbon number and double bond equivalents between samples.

DigiFab Interacts With Endogenous Cardiotonic Steroids and Reverses Preeclampsia-Induced Na/K-ATPase Inhibition

Elevated levels of endogenous Na/K-ATPase (NKA) inhibitors, cardiotonic steroids (CTSs) including marinobufagenin (MBG), contribute to pathogenesis of preeclampsia (PE) and represent a target for immunoneutralization by Digibind (Ovine Digoxin Immune Antibody, Glaxo-Smith Kline). Because Digibind is no longer commercially available, we studied whether DigiFab (BTG International Ltd, UK) can substitute Digibind for immunoneutralization of CTS in patients with PE. We compared DigiFab, Digibind, and anti-MBG monoclonal antibody (mAb) with respect to their ability to interact with CTS in PE plasma and to restore NKA activity in erythrocytes from patients with PE. Using immunoassays based on DigiFab, Digibind, and anti-MBG mAb, we studied the elution profile of CTS following high-performance liquid chromatography (HPLC) fractionation of PE plasma. Totally, 7 patients with mild PE (28 ± 2 years; gestational age, 39 ± 0.5 weeks; blood pressure 156 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant participants (28 ± 1 years; gestational age, 39 ± 0.4 weeks; blood pressure 111 ± 2/73 ± 2 mm Hg) were enrolled. Preeclampsia was associated with a substantial inhibition of erythrocyte NKA (1.47 ± 0.17 vs 2.65 ± 0.16 µmol Pi/mL per h in control group, P < .001). Ex vivo, at 10 µg/mL concentration, which is consistent with the clinical dosing of Digibind administered previously in PE, DigiFab and Digibind as well as anti-MBG mAb (0.5 µg/mL) restored erythrocyte NKA activity. Following HPLC fractionation of pooled PE and control plasma, PE-associated increase in CTS material was detected by Digibind (176 vs 75 pmoles), DigiFab (221 vs 70 pmoles), and anti-MBG mAb (1056 vs 421 pmoles). Therefore, because DigiFab interacts with CTS from PE plasma and reverses PE-induced NKA inhibition, it can substitute Digibind for immunoneutralization of CTS in patients with PE.

WYE‐120318, a ring contraction product of methylnaltrexone, and structure revision of coniothyrione

A contracted ring degradation product, WYE-120318 (compound 2), was discovered during the development phase for methylnaltrexone bromide (compound 1) drug substance. The compound was isolated by high-performance liquid chromatography fractionation, and its structure was determined by spectroscopic data analyses. WYE-120318 is formed from methylnaltrexone through a benzyl-benzilic acid type rearrangement reaction to yield an α-hydroxy-cyclopentanecarboxylic acid substructure. The proposed structure and the formation mechanism are confirmed by the synthesis of WYE-120318 from methylnaltrexone (compound 1). A similar benzyl-benzilic acid type rearrangement reaction can be envisioned as the biological origin of remisporine A (compound 3), a naturally occurring cyclopentadienyl compound that autocatalytically dimerizes to remisporine B (compound 4). The structure of remisporine A was deduced from its dimer 4. Coniothyione (compound 5) can be considered as the first example of a stable natural product bearing the remisporine A skeleton. However, the regiochemistry of the chlorosubstitution in the coniothyrione structure needs to be revised to compound 6 on the basis of the nuclear magnetic resonance data and biogenesis analysis.

Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus)

The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0–4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at −20°C is not feasible. The two cheetahs in Germany also received an injection of [3H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs.

Immunological determination of gliadin 33‐mer equivalent peptides in beers as a specific and practical analytical method to assess safety for celiac patients

Cereals used for beer manufacturing contain gluten, which is immunotoxic for celiac patients. The gluten remaining after processes of malting and brewing is mostly hydrolyzed, which makes practical evaluation of the immunotoxicity of the gluten pools challenging. We analyzed the presence of gluten peptides equivalent to the major immunotoxic protease-resistant gliadin 33-mer in 100 Belgium beers, using monoclonal antibodies (G12/A1). Immunochromatographic strips and enzyme-linked immonosorbent assay G12/A1 methods estimated at least 20 ppm gluten equivalents in 90 beers and gluten-free in 10 beers. The G12/A1 reactivity of beer high-performance liquid chromatographic fractions correlated to the presence of T-cell-reactive epitopes identified by peptide sequencing. The determination of equivalent gliadin 33-mer epitopes in beers has been shown to be practical, specific, and sensitive for the measurement of potential immunotoxicity for celiac patients.

Reuse of drinking water treatment sludge for olive oil mill wastewater treatment

Olive mill wastewater (OMW) results from the production of olive oil, which is an important traditional agro-industry in Mediterranean countries. In continuous three-phase centrifugation 1.0-1.2 m(3) of OMW are produced per ton of processed olives. Discharge of OMW is of serious environmental concern due to its high content of organic matter with phytotoxic properties, namely phenolic compounds. Meanwhile, drinking water treatment sludge (DWTS) is produced in high amounts and has long been considered as a waste for landfill. The aim of this work was the assessment of reusing DWTS for OMW treatment. High performance liquid chromatography (HPLC) analysis was carried out to determine the phenolic compounds present and to evaluate if they are recalcitrant. Treatability assays were performed using a dosage of DWTS from 50 to 300 g L(-1). Treatment efficiency was evaluated based on the removal of chemical oxygen demand (COD), biochemical oxygen demand (BOD), total solids (TS), total suspended solids (TSS), total volatile solids (TVS), oil and grease (OG), phenols (total phosphorous (TP) and HPLC fraction). Results from OMW HPLC characterization identified a total of 13 compounds; the major ones were hydroxytyrosol, tyrosol, caffeic acid, p-cumaric acid and oleuropein. Treatability assays led to a maximum reduction of about 90% of some of the phenolic compounds determined by HPLC. Addition of 200-300 g L(-1) of DWTS reduced 40-50% of COD, 45-50% of TP, a maximum of nearly 70% TSS and 45% for TS and TVS. The OG fraction showed a reduction of about 90%, achieved adding 300 g L(-1) od DWTS. This study points out the possibility of establishing an integrated management of OMW and DWTS, contributing to a decrease in the environmental impact of two industrial activities, olive oil production and drinking water treatment.

Sample handling and contamination encountered when coupling offline normal phase high performance liquid chromatography fraction collection of petroleum samples to Fourier transform ion cyclotron resonance mass spectrometry

Normal phase high performance liquid chromatography (HPLC) is used to separate a gas oil petroleum sample, and the fractions are collected offline and analyzed on a high resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR MS). The separation prior to MS analysis dilutes the sample significantly; therefore the fractions need to be prepared properly to achieve the best signal possible. The methods used to prepare the HPLC fractions for MS analysis are described, with emphasis placed on increasing the concentration of analyte species. The dilution effect also means that contamination in the MS spectra needs to be minimized. The contamination from molecular sieves, plastics, soap, etc. and interferences encountered during the offline fraction collection process are described and eliminated. A previously unreported MS contamination of iron formate clusters with a 0.8 mass defect in positive mode electrospray is also described. This interference resulted from the stainless steel tubing in the HPLC system. Contamination resulting from what has tentatively been assigned as palmitoylglycerol and stearoylglycerol was also observed; these compounds have not previously been reported as contaminant peaks.

Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in spotted hyenas (Crocuta crocuta)

The use of enzyme immunoassays (EIAs) to measure faecal glucocorticoid metabolites (fGCM) is a useful non-invasive technique to monitor adrenocortical activity in vertebrates. The first objective of this study was to validate an ‘in-house’ EIA (cortisol-3-CMO) for the measurement of fGCM concentrations in spotted hyenas. High-performance liquid chromatography (HPLC) was used to characterise fGCM in samples from a captive hyena that received an i.v. injection of [3H] cortisol. All HPLC fractions were analysed with the EIA for the presence and quantities of radiolabelled fGCM. Radiolabelled fGCM consisted of substances with a higher polarity than cortisol and substances of lower polarity that eluted between cortisol and corticosterone. Authentic radiolabelled cortisol was not detected. The EIA measured substantial amounts of immunoreactivity corresponding to the radioactive peaks. It also detected a significant increase in fGCMs after an adrenocorticotropic hormone (ACTH) challenge in two other captive animals and a significant increase in fGCMs in a fourth captive animal after anaesthesia. The second objective was to investigate an age effect on fGCM: we conducted pairwise comparisons of fGCM concentrations in individual free-ranging juvenile spotted hyenas when less than 6months of age and when between 6 and 24months of age. We expected juveniles to experience a more unpredictable and therefore more stressful environment when younger than when older. When younger, juveniles had significantly higher fGCM concentrations than when they were older. Our results demonstrate that our assay can be used to assess adrenocortical activity in spotted hyenas.

Characterization of organic matter in alum treated drinking water using high performance liquid chromatography and resin fractionation

In order to understand and improve drinking water treatment process operations, many techniques have been established to characterize natural organic matter (NOM). Resin fractionation is the most widely used technique to isolate NOM based on its hydrophobicity and hydrophilicity, these can be used to determine the treatability of NOM, however, it is also recognized as a time consuming technique. This paper describes the use of reverse phase high performance liquid chromatography (RPHPLC) as a rapid assessment of the hydrophobicity/hydrophilicity of NOM. The reduction of total RPHPLC peak area correlated well with dissolved organic carbon (DOC) and UV absorbance at 254nm (UV254) removal efficiency. After resolving the RPHPLC profile using a peak fitting technique, the ratio between hydrophobic and hydrophilic peak area can be used to quantify the treatability of NOM (particularly DOC removal). Further statistical analysis grouped the resolved peaks into three groups (two groups of hydrophilic and one group of hydrophobic) and established an expression to link peak area with UV254 and DOC removal. The characterization results were further compared by the traditional resin fractionation technique using DAX-8 and XAD-4 resins (combined). The results confirmed that despite the definition that hydrophobic and hydrophilic components measured by both methods could be different, both methods confirm that the hydrophilic fraction was recalcitrant after coagulation and is of small molecular weight.

A metallomics approach discovers selenium-containing proteins in selenium-enriched soybean

Our previous study found that high-molecular-weight selenium (Se) species make up 82% of the total Se in the bean of Se-enriched soybean plants (Chan et al. 2010, Metallomics, 2(2): p. 147-153). The Se species have been commonly seen in other plants in addition to soybean, but their identities remain unresolved. The present study employs a multi-technique metallomics approach to characterize the proteins containing Se in the beans of Se-enriched soybean plants. Two main categories of proteins, maturation proteins and protease inhibitors, were found in Se-containing high-performance liquid chromatography (HPLC) fractions. The proteins were screened by two-dimensional HPLC-inductively coupled plasma mass spectrometry, size-exclusion chromatography, and anion-exchange chromatography, and the Se-containing fractions were then identified by peptide mapping using HPLC-Chip-electrospray ion trap mass spectrometry. Based on the belief that Se goes into proteins through non-specific incorporation, a new method was designed and applied for the Se-containing peptide identification. The Se-containing peptide KSDQSSSYDDDEYSKPCCDLCMCTRS, part of the sequence of protein Bowman-Birk proteinase isoinhibitor (Glycine max), was found in one of the Se-containing fractions. The nutritional value of the Se-containing proteins in Se-enriched soybeans will be an interesting topic for the future studies.

Identification of new lignans in Norway spruce knotwood extracts

Abstract In a hydrophilic extract of Norway spruce knotwood, the dominating lignan, 7-hydroxymatairesinol, was partially removed by precipitation, and the resulting mixture was fractionated by flash chromatography and preparative high-performance liquid chromatography (HPLC). In the HPLC fractions, 7S- and 7R-todolactol A, 7′-hydroxylariciresinol, and two stereoisomers of 9′-hydroxylariciresinol were identified by gas chromatography-mass spectrometry (GC-MS) analyses, and their structures were confirmed by nuclear magnetic resonance spectroscopy. The 9′-hydroxylariciresinols were suggested to have the 7S,8R,8′R,9′Rand 7R,8R,8′R,9′Rconfigurations, and (-)-7′-hydroxylariciresinol 7S*,7′R*,8R*,8′S*, which indicates a configuration of this structure that has not been reported previously. 7S- and 7R-isoliovil were identified by comparison with previously published GC-MS data, and 7′-oxolariciresinol was tentatively identified on the basis of its mass spectrum. Of these lignans, only 7′-hydroxylariciresinol has been identified previously in Norway spruce. Several other lignans with similar mass spectra as the todolactols, isoliovils, and 7′-hydroxylariciresinol were detected, indicating that they are different stereoisomers of these lignans and/or of liovils. In addition to these lignans, the mass spectra of several other unidentified minor lignans indicated the presence of several tens of previously unidentified minor lignans in Norway spruce knotwood, accounting altogether for approximately 6% of the dry weight of the ethanol extract. 7S-Todolactol A, which was dominating among the new lignans, was found to be very unstable in aqueous solutions. Identification of more of these unidentified lignans may be possible only by access to pure reference compounds.

The combination of analytical-scale HPLC separation with a TR-FRET assay to investigate JAK2 inhibitory compounds in a Boysenberry drink

We report the detection of JAK2 inhibitory activity in a Boysenberry (Rubus loganbaccus x R. baileyanus Britt.) drink using a combination of analytical-scale high performance liquid chromatography (HPLC) with a high-sensitivity time-resolved fluorescence coupled with fluorescence resonance energy transfer (TR-FRET) method. Phytochemical components of a Boysenberry drink were separated by reversed phase HPLC , and 84 separate fractions were collected. HPLC fractions corresponding to the ellagitannin and ellagic acid peaks observed in the chromatogram inhibited JAK2 activity. Anthocyanins, while they were the major phytochemical components of the Boysenberry drink, had no JAK2 inhibitory activity even though anthocyanins have previously been shown to be anti-inflammatory. This study demonstrates the usefulness of combining rapid analytical-scale HPLC separation with a highly sensitive fluorescence bioassay for characterising bioactivity in complex plant extracts. Ellagic acid was found to have an IC(50) of 92 nM against JAK2 and complete inhibition of JAK2 activity was observed in HPLC fractions of Boysenberry extract which had been diluted several hundred fold. To the best of our knowledge, this is the first demonstration that ellagitannins and other natural ellagic acid analogues are potent inhibitors of JAK2. Thus a drink containing Boysenberry juice concentrate may have anti-inflammatory properties.

Analysis of co-eluted isomers of high-molecular weight polycyclic aromatic hydrocarbons in high performance liquid chromatography fractions via solid-phase nanoextraction and time-resolved Shpol'skii spectroscopy

We present an accurate method for the determination of isomers of high-molecular weight polycyclic aromatic hydrocarbons co-eluted in HPLC fractions. The feasibility of this approach is demonstrated with two isomers of molecular weight 302 with identical mass fragmentation patterns, namely dibenzo[ a,i]pyrene and naphtho[2,3- a]pyrene. Qualitative and quantitative analysis is carried out via laser-excited time-resolved Shpol'skii spectroscopy at liquid helium temperature. Unambiguous identification of co-eluted isomers is based on their characteristic 4.2 K line-narrowed spectra in n-octane as well as their fluorescence lifetimes. Pre-concentration of HPLC fractions prior to spectroscopic analysis is performed with the aid of gold nanoparticles via an environmentally friendly procedure. In addition to the two co-eluted isomers, the analytical figures of merit of the entire procedure were evaluated with dibenzo[ a,l]pyrene, dibenzo[ a,h]pyrene and dibenzo[ a,e]pyrene. The analytical recoveries from drinking water samples varied between 98.2 ± 5.5 (dibenzo[ a,l]pyrene) and 102.7 ± 3.2% (dibenzo[ a,i]pyrene). The limits of detection ranged from 51.1 ng L −1 (naphtho[2,3- a]pyrene) to 154 ng L −1 (dibenzo[ a,e]pyrene). The excellent analytical figures of merit associated to its HPLC compatibility makes this approach an attractive alternative for the analysis of co-eluted isomers with identical mass spectra.

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1⁻/⁻) mice were used. Apoa1⁻/⁻ mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1⁻/⁻ mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1⁻/⁻ mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.

Proteomic Analysis of Centruroides tecomanus (Buthidae scorpion) Venom and Biological Activity of Venom Fractions

Centruroides tecomanus is a Mexican scorpion of the state of Colima. It is a medically important scorpion, but its venom is poorly studied. Here we report the separation of the soluble venom of this scorpion by high‐performance liquid chromatography (HPLC). From 98 fractions obtained, 50 distinct components were identified by electrospray mass spectrometry (LC/ESI‐MS) analysis. The molecular masses distribution of these components varies from 904 to 7555 Da, from which 48% falls in between 5500–7555 Da, 38% within the range of 3000–5500 Da and 12% within the range 900–3000 Da. The biological effects of the venom were tested on crustacean/freshwater shrimp (Cambarellus montezumae), insects/cockroach (Acheta domestica) and mammals/mouse (strain CD1). For the bioassays the HPLC fractions obtained during 60 minutes run were separated into 12 groups (5 min elution time each). Group VII was lethal to the three species used for assay. The IV group had toxic effect in freshwater shrimps whereas groups VI, VII and VIII were all lethal. For cockroach, groups V and VI were toxic and group VII was lethal. In mouse the lethal components were found in group VII; group VIII was toxic. The molecular masses in the mouse lethal group fall between 7013 and 7487 Da. Two lethal peptides for the three kinds of animals used, they were eluted from the HPLC at 32.72 minutes with molecular weight of 6334 Da and 31.85 min with 7430 Da, respectively.