Spin-trapped Radicals: Determination by LC-TSP-MS and LC-ESI-MS

The 4-POBN[α-(4-pyridyl-l-oxide)-N-tert-butyl-nitrone] radical adducts of ethyl and pentyl radicals were determined by a combination of high performance liquid chromatography (HPLC) combined with electron paramagnetic resonance (EPR) with HPLC-electrospray (ESI)-mass spectrometry and HPLC-thermospray (TSP)-MS. The identifIcation of the peak corresponding to the spin-trapped radical was done by performing HPLC-EPR under the same chromatographic conditions as the HPLC-MS. The radical adducts could be determined by both techniques, even though for ESI only 12 μL/min of the total 1 mL/min HPLC flow rate could be directed into the ion source.     

Investigation of enhanced ion abundances from a carrier process in high-performance liquid chromatography particle beam mass spectrometry

Enhancements in ion abundances in high-performance liquid chromatography (HPLC) particle beam (PB) mass spectrometry were observed during coelution or when certain substances (e.g., ammonium acetate) were added to the mobile phase during a reverse-phase gradient elution HPLC separation. These enhancements were observed with two commercial PB interfaces and a variety of compounds generally not amenable to separation by gas chromatography. The enhancements are attributed to both improved chromatographic efficiency and a PB carrier process.     

Neutral Serine Protease from Penicillium italicum. Purification,Biochemical Characterization,and Use for Antioxidative Peptide Preparation from Scorpaena notata Muscle

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0–9.0. The protease was activated by divalent cations such as Ca²⁺ and Mg²⁺. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.     

Rapid and Sensitive LC–MS/MS Analysis of Fatty Acids in Clinical Samples

Free fatty acids (FFAs), major cellular metabolites, play an important role during tumor pathogenesis. Enhanced de novo fatty acid synthesis in tissues is a characteristic feature of cancer. Therefore, measurement of FFA concentration in biological samples is beneficial for cancer research and clinical diagnosis. Herein, a rapid, stable, and sensitive detection methodology was established to simultaneously quantify 22 FFAs using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS). The HPLC–MS/MS system was run in negative ion mode for 15 min using multiple reaction monitoring. The lipids were extracted from colon tissues of colon cancer patients and then injected into the HPLC–MS/MS system for analysis. Colon samples were analyzed by inter-day repeatability and intra-day repeatability, with less than 5 % deviation for most fatty acids. This approach is successful to determine low picogram concentrations of each FFA molecule using milligrams of tissue, and provides a promising method for FFA microanalysis in clinical samples.     

Determination of neurotransmitters and their metabolites using one- and two-dimensional liquid chromatography with acidic potassium permanganate chemiluminescence detection

High-performance liquid chromatography with chemiluminescence detection based on the reaction with acidic potassium permanganate and formaldehyde was explored for the determination of neurotransmitters and their metabolites. The neurotransmitters norepinephrine and dopamine were quantified in the left and right hemispheres of rat hippocampus, nucleus accumbens and prefrontal cortex, and the metabolites vanillylmandelic acid, 3,4-dihydrophenylacetic acid, 5-hydroxyindole-3-acetic acid and homovanillic acid were identified in human urine. Under optimised chemiluminescence reagent conditions, the limits of detection for these analytes ranged from 2.5?×?10^?8 to 2.5?×?10^?7 M. For the determination of neurotransmitter metabolites in urine, a two-dimensional high-performance liquid chromatography (2D-HPLC) separation operated in heart-cutting mode was developed to overcome the peak capacity limitations of the one-dimensional separation. This approach provided the greater separation power of 2D-HPLC with analysis times comparable to conventional one-dimensional separations. Figure

2D-HPLC separation and permanganate chemiluminescence detection of neurotransmitter metabolites     

Melt miscibility of HDPE/UHMWPE,LDPE/UHMWPE,and LLDPE/UHMWPE blends detected by dynamic rheometer

The dynamic rheological behavior of high density polyethylene (HDPE)/ultrahigh molecular weight polyethylene (UHMWPE) blends, low density polyethylene (LDPE)/UHMWPE blends and linear low density polyethylene (LLDPE)/ UHMWPE blends was measured in parallel plate rheometer at 200°C. The analysis of log-additivity rule, Cole-Cole plots and Han curves of the three series blends indicated that the LDPE/UHMWPE blends were miscible in the melt, while the HDPE/UHMWPE blends and LLDPE/UHMWPE blends showed phase separation. The DSC results of LLDPE/UHMWPE blends and HDPE/UHMWPE blends were consistent with the rheological properties, while for the thermal properties of LDPE/UHMWPE blends, results revealed three endothermic peaks, which indicated a liquid-solid phase separation in LDPE/UHMWPE blends.     

Production of Pt nanoparticles-supported chelating group-modified graphene for direct methanol fuel cells

In this study, Pt nanoparticles (NPs) were supported on reduced graphene oxide with the aid of disodium ethylenediamine-tetraacetate, where the Pt iona were initially attached to EDTA-functionalized graphene oxide (EDTA-GO) sheets and then the metal ion and the graphene oxide were reduced simultaneously by ethylene glycol. Electrochemical properties of the catalysts were studied by measuring cyclic voltammetries, and functional groups of the synthesized materials were investigated by Fourier transform infrared spectrometry. Average sizes and lattice parameters were measured by scanning electron microscopy, transmission electron microscopy images, and X-ray diffraction. The results showed that Pt NPs were successfully deposited on the EDTA-GO with the crystallite size of about 2.3 nm. The prepared catalysts demonstrated an enhanced tolerance towards CO poisoning, when EDTA-GO was used as supports. This suggests that EDTA plays a crucial role in the dispersion and electrocatalytic activity of the metal nanoparticles.     

Ultrasensitive detection and identification of BRAF V600 mutations in fresh frozen,FFPE, and plasma samples of melanoma patients by E-ice-COLD-PCR

A number of molecular diagnostic methods have been developed for the detection and identification of mutations in tumor samples, which are important for the choice of treatment in the context of personalized medicine. For the treatment of metastatic melanoma, Vemurafenib is recommended for patients with BRAF V600 activating mutations. However, the different assays developed to date for the detection of these mutations lack sensitivity or specificity or do not allow a sequencing-based identification or validation of the mutation. Recently, enhanced improved and complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) has been developed as a sensitive method for the detection and identification of mutations in KRAS codons 12/13. Here, we present the first E-ice-COLD-PCR assay for the detection and identification of BRAF codon 600 mutations, which has a large dynamic range, as 25 pg to 25 ng can be used as DNA input without any reduction in mutation enrichment efficiency, and which can detect down to 0.01 % of mutated alleles in a wild-type background. The assay has been validated on fresh frozen, formalin-fixed paraffin-embedded (FFPE), and plasma samples of melanoma patients and has allowed the detection and identification of BRAF mutations present in samples appearing as wild type using standard pyrosequencing, endpoint genotyping, or Sanger sequencing. Thus, the BRAF V600 E-ice-COLD-PCR assay is currently one of the most powerful molecular diagnostic tools for the ultrasensitive detection and identification of BRAF codon 600 mutations.     

Synthesis and preparation of nanoparticles composed of amphiphilic poly(γ-glutamic acid) with different hydrophobic side chains and their potential of membrane disruptive activity

In the development of nanoparticle-based vaccine adjuvants, the interaction between nanoparticles (NPs) and the cells is a key factor. To control them, we focused on the relationship between the hydrophobicity of the side chains and the cell membrane. In this study, amphiphilic poly(γ-glutamic acid) (γ-PGA), using various types of hydrophobic side chains, was synthesized and used to prepare NPs for evaluating the membrane disruptive activity. When leucine ethyl ester (Leu), methionine ethyl ester (Met), or tryptophan ethyl ester (Trp) was grafted, each polymer formed monodispersed NPs at physiological conditions. Significantly, NPs composed of Leu and Trp showed a membrane disruptive activity at the endosomal environment (pH 5–6.5), while NPs composed of Met did not show. This might be due to the weak hydrophobicity of Met compared to that of Leu and Trp, which demonstrated that the interaction between NPs and cells could be controlled by designing the polymer compositions.     

Multiresidue Determination of Ten Nonsteroidal Anti-inflammatory Drugs in Bovine,Porcine, and Chicken Liver Tissues by HPLC-MS/MS

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the group of drugs having the therapeutic efficacy of analgesic and antipyretic. To detect health-threatening residues of NSAIDs, a fast and easy multiresidue method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) was described. Ten NSAIDs were extracted from the tissues using 2 mL of acetonitrile and 0.1 mL of 2 mM ammonium formate in distilled water. After clean-up using C18 sorbent, it was evaporated under nitrogen, reconstituted with 1 mL distilled water and analyzed by LC-MS/MS. The method was validated based on guideline for residue testing laboratory. Furthermore, the method has also been applied successfully to detect ten NSAIDs from bovine, porcine, and chicken liver tissues. In a total of 315 liver samples tested, acetylic salicylic acid was detected from 28 porcine and 2 chicken liver tissues at levels of 13?～?576 and 50?～?53 ng/g, respectively. Subsequently, paracetamol was detected in 15 porcine liver tissues with a detection levels of 28?～?381 ng/g. Phenylbutazone and its metabolite, oxyphenylbutazone, were detected at 247 and 15 ng/g range in one of the bovine liver tissue, respectively.     

New EDTA determination method based on ion chromatography with suppressed conductimetric detection

A novel direct method for the determination of EDTA in alkaline radioactive evaporator residue solution was developed and validated based on ion chromatography with suppressed conductimetric detection and anion exchange columns (A Supp 4, 4 mm × 250 mm and A Supp 5, 4 mm × 150 mm). The yttrium-EDTA complex resulted one single chromatographic peak in the eluent and allowed the correct determination of EDTA in an alkaline, high concentration radioactive waste water. Depending on coexisting substances, suitable eluent is 10.0 mM carbonate buffer/pH 10.6 or 10.75 (t _R,Y–EDTA = 7.01 and 6.4 min, respectively). For 10.0 mM carbonate buffer/pH 10.6 and isocratic flow rate of 1.0 cm³/min, a linear calibration curve was obtained from 5 to 40 mg/dm³ (r > 0.999) EDTA. Good resolution was achieved from commonly coexisting anions (chloride, nitrite, nitrate, sulphate, phosphate, bromide and citrate). The developed simple ion chromatographic method was applied for the assay of EDTA in various radioactive alkaline solutions.     

Development of ssDNA Aptamers for the Sensitive Detection of Salmonella typhimurium and Salmonella enteritidis

Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica subsp. enterica ser. typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. The development of an early and ultra-sensitive detection system is the first critical step in controlling this disease. To accomplish this, we used the cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. A total of 12 target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX under stringent selection conditions, and negative selection further enhanced the selectivity among these aptamers. Aptamer specificity was investigated using the gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar to submicromolar levels. Furthermore, individual aptamers were conjugated onto polyvalent directed aptamer polymer, which led to 100-fold increase in binding affinity compared to the individual aptamers alone. Taken together, this study reports the identification of higher affinity and specificity ssDNA aptamers (30mer), which may be useful as capture and detection probes in biosensor-based detection systems for salmonellosis.     

A colorimetric assay for d-Penicillamine in urine and plasma samples based on the aggregation of gold nanoparticles

We report herein the development of a highly sensitive colorimetric method for detection of d-Penicillamine using citrate-capped gold nanoparticles (AuNPs). This assay relies upon the distance-dependent of gold nanoparticles surface plasmon resonance band of gold nanoparticles. By replacing the thiol-containing chelator drug, d-Penicillamine, with citrate on the gold nanoparticles surface, a new peak appearing at a longer wavelength intensifies and shifts further to the red from the original peak position due to aggregation of gold nanoparticles which depends on ionic strength, gold nanoparticles and d-Penicillamine concentration. During this process, the plasmon band at 521 nm decreases gradually along with the formation of a new red-shifted band at 630 nm. The calibration curve which is derived from the ratio intensities of absorbance at longer wavelength (630 nm) to original wavelength (521 nm) displays a linear relation in the range of 5.0 × 10^?6–3.0 × 10^?4 M d-Penicillamine. Lower limit of detection for d-Penicillamine, at the signal-to-noise ratio of 3 (3σ), was 3.8 × 10^?6 M. The developed methodology was successfully applied for the determination of d-Penicillamine in human urine and plasma.     

The Prognostic Value of Histidine-Rich Glycoprotein RNA in Breast Tissue Using Unmodified Gold Nanoparticles Assay

The aim of is this study is to explore the role of tissue histidine-rich glycoprotein (HRG) RNA as a promising clinically useful biomarker for breast cancer patients prognosis using nanogold assay. Expression of the HRG RNA was assessed by gold nanoparticles and conventional RT-PCR after purification by magnetic nanoparticles in breast tissue samples. The study included 120 patients, 60 of which were histologically proven breast carcinoma cases, 30 had benign breast lesions and 30 were healthy individuals who had undergone reductive plastic surgery. ER, PR and HER2 status were also investigated. The prognostic significance of tissue HRG RNA expression in breast cancer was explored. The magnetic nanoparticles coated with specific thiol modified oligonucleotide probe were used successfully in purification of HRG RNA from breast tissue total RNAs with satisfactory yield. The developed HRG AuNPs assay had a sensitivity and a specificity of 90 %, and a detection limit of 1.5 nmol/l. The concordance rate between the HRG AuNPs assay with RT-PCR after RNA purification using magnetic nanoparticles was 93.3 %. The median follow-up period was 60 months. Among traditional prognostic biomarkers, HRG was a significant independent prognostic marker in relapse-free survival (RFS). HRG RNA is an independent prognostic marker for breast cancer and can be detected using gold NPs assay, which is rapid, sensitive, specific, inexpensive to extend the value for breast cancer prognosis.     

Efficient Production of Native Lunasin with Correct N-terminal Processing by Using the pH-Induced Self-Cleavable Ssp DnaB Mini-intein System in Escherichia coli

To develop an efficient and cost-effective approach for the production of small preventive peptide lunasin with correct natural N terminus, a synthetic gene was designed by OPTIMIZER & Gene Designer and cloned into pTWIN1 vector at SapI and PstI sites. Thus, lunasin was N-terminally fused to the pH-induced self-cleavable Ssp DnaB mini-intein linked to a chitin binding domain (CBD) with no extra residues. The resultant fusion protein was highly expressed by lactose induction in Escherichia coli BL21 (DE3) in a 7-l bioreactor and bound to a chitin affinity column. After washing the impurities, the Ssp DnaB intein mediated on-column self-cleavage was easily triggered by shifting pH and temperature to allow the native lunasin released. The final purified lunasin yielded up to 75 mg/l medium. Tricine/SDS-PAGE and matrix-assisted laser desorption time-of-flight (MALDI-TOF)/mass spectrometry (MS) verified the structural authenticity of the product, implying the correct cleavage at the junction between Ssp DnaB intein and lunasin. MTT assay confirmed its potent proliferation inhibitory activity to human cancer cells HCT-116 and MDA-MB-231; however, no cytotoxicity to normal human lens epithelial cell SRA01/04 and hepatoma HepG2. Taken together, we provide a novel strategy to produce recombinant native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein.     

Low surface area carbonized silica obtained by the pyrolysis of dichloromethane: A column packing for adsorption and exclusion chromatography

Carbonized silica has been used as column packing for HPLC. Up to now it has been obtained by the pyrolysis of the vapour of benzene as well as aliphatic and aromatic alcohols. This paper reports on the properties of carbonized silica obtained by the pyrolysis of dichloromethane and used as column packing in HPLC.     

Quantitative analysis by high-performance liquid chromatography atmospheric pressure chemical ionization mass spectrometry: The determination of the renin inhibitor CP-80,794 in human serum

A specific method was developed for the quantitative determination of the renin inhibitor CP-BO,794. Serum extracts containing the drug and an internal standard were injected into a standard reverse-phase high-performance liquid chromatography (HPLC) column. The mobile phase, methanol/water (8/2), flowed at 1 ml/min through the column and then via a heated nebulizer interface into a corona discharge atmospheric pressure chemical ionization source. The assay minimum limit of quantification was 50 pg/mL. It exhibits satisfactory accuracy and precision over the range 50 pg/ml, to 10 ng/mL. A minor modification of the HPLC mobile phase was necessary to attain extremely low detection limits. The addition of a structural analogue contributed to enhancing the precision of the assay.     

Solid-phase methylamidation for sialoglycomics by MALDI-MS

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been a major approach for glycan analysis. However, the preferential cleavage of the sialic acid moiety by in- and post-source decay influences the determination of sialylated glycans by MALDI-MS. Many chemical derivatization methods were introduced to stabilize the sialylated glycan during MALDI-MS. Among current derivatization methods, methylamidation is a promising means for simultaneous analysis of natural sialylated glycans regardless of their sialic acid linkage types. Here, a novel derivatization method was developed, in which proteins were conjugated on the solid-phase support in order to stabilize the sialic acids by methylamidation and to reduce sample loss and contamination during the derivatization process. This derivatization strategy was used to investigate N-glycans from fetuin, a glycoprotein containing different types of complex N-glycans. The developed method was also applied to the N-glycan profiling of human serum from patients and healthy volunteers. Results were consistent with N-glycan profiling by HPLC-fluorescence detection. This new method provided a sensitive, simple, and robust approach for profiling glycan structures of complex samples.