Determination of rat serum esterase activities by an HPLC method using S-acetylthiocholine iodide and p-nitrophenyl acetate

Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the ¹⁴C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.     

High-performance liquid chromatographic analysis of nitrite and nitrate in human plasma as S-nitroso-N-acetylcysteine with ultraviolet absorbance detection

A rapid HPLC method with UV absorbance detection at 333 nm for the measurement of nitrite and nitrate in ultrafiltrate samples of human plasma is described. The method is based on hydrochloric acid-catalyzed conversion of nitrite by N-acetyl-l-cysteine to S-nitroso-N-acetyl-l-cysteine and isocratic elution using 10 mM NaH₂PO₄ in acetonitrile–water, pH 2.0 (15:85, v/v). The limit of detection of the method is 50 nM nitrite. The method was validated by gas chromatography–mass spectrometry.     

Methyl malondialdehyde as an internal standard for the determination of malondialdehyde

Methyl malondialdehyde (Me-MDA) is suggested as an internal standard for the determination of the lipid peroxidation product, malondialdehyde (MDA). A procedure for synthesising the Me-MDA sodium salt is described in detail. The purity and identity of the synthesised Me-MDA have been confirmed using nuclear magnetic resonance and UV spectroscopy, and by micellar electrokinetic chromatography. The applicability of Me-MDA as an internal standard has been demonstrated for rat brain homogenate samples. These samples were purified solely through ultrafiltration. The preferred analytical technique was capillary zone electrophoresis (CZE) with UV detection at 267 nm. The limits of detection (3 S/N) for the CZE separations of Me-MDA and MDA were 0.5 and 0.2 μM, respectively, and the total analysis time was approximately 10 min. Details of separations are also presented using high-performance liquid chromatography (HPLC) with UV detection at 245 nm, and gas chromatography, together with either electron capture or mass spectrometric detection. The GC separations require derivatisation of MDA and Me-MDA with pentafluorophenylhydrazine while the CZE and HPLC separations can be performed on the native molecules.     

Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene

Screening for large gene rearrangements is established as an important part of molecular medicine but is also challenging. A variety of robust methods can detect whole-gene deletions, but will fail to detect more subtle rearrangements that may involve a single exon. In this paper, we describe a new, versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography assay (MP/LC). Multiple exons are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and quantitated by fluorescent detection using a post-column intercalation dye. The relative peak intensities for each target directly reflect exon copy number. This novel technique was used to screen a panel of 121 unrelated retinoblastoma patients who were tested previously using a reference strategy. MP/LC correctly scored all deletions and demonstrated a previously undetected RB1 duplication, the first to be described. MP/LC appears to be an easy, versatile, and cost-effective method, which is particularly relevant to denaturing HPLC (DHPLC) users since it broadens the spectrum of available applications on a DHPLC system.     

Extraction and quantification of nicardipine in human plasma

A novel simple method of extraction, separation, identification and quantification of nicardipine in human plasma samples was completely studied. The human plasma samples were initially purified by solid-phase extraction (SPE) using a C₁₈ cartridge. The extracted samples were separated and nicardipine present in the samples was quantified by high-performance liquid chromatography (HPLC) on a reversed-phase C₁₈ column employing a mobile phase consisting of 60% (v/v) acetonitrile in 0.02 M NaH₂PO₄ with pH of 6.3 and a variable wavelength UV detector set at 254 nm. The recovery of nicardipine from plasma samples using selective SPE was 91±6.0% and had less interfering compounds in the HPLC analysis compared to the use of liquid–liquid (L/L) extraction. In the HPLC analysis, examining the effect of pH values of the mobile phase on the capacity factor (k′) of nicardipine revealed a method for selecting a critical k′ value of nicardipine to eliminate interfering peaks near the peak specific to the analyte. This method for quantification of nicardipine in human plasma samples was suitable for studying the pharmacokinetic profile of nicardipine administered as an intravenous bolus to cardiac surgical patients.     

Ultraviolet and tandem mass spectrometry for simultaneous quantification of 21 pivotal metabolites in plasma from patients with diabetic nephropathy

A sensitive and specific method was developed for simultaneous determination of 21 compounds related to the diabetic nephropathy (DN) in a single analysis using high-performance liquid chromatography coupled to ultraviolet and tandem mass spectrometry (HPLC–UV/MS/MS) in human plasma. With retention times and MS/MS for peak identification, both UV and MS detectors were used for quantification. Calibration curves suitable for the analysis of plasma were linear (r² > 0.998) with limits of detection (LOD) from 10 to 1000 ng/mL. Intraday relative standard deviation (R.S.D.) and interday R.S.D. were both lower than 15%. With the case and control study, we found five potential biomarkers of DN, including adenosine, inosine, uric acid, xanthine and creatinine.     

Antimicrobial Properties of an Oxidizer Produced by Burkholderia cenocepacia P525

A compound with both oxidizing properties and antibiotic properties was extracted and purified from broth cultures of Burkholderia cenocepacia strain P525. A four step purification procedure was used to increase its specific activity ~400-fold and to yield a HPLC–UV chromatogram containing a single major peak. Size exclusion chromatography suggests a molecular mass of ~1,150 and UV spectroscopy suggests the presence of a polyene structure consisting of as many as six conjugated double bonds. Biological studies indicate that the compound is bacteriostatic. Enterobacter soli and E. aerogenes cells incubated with the compound exhibit a longer lag phase of growth. The bacteriostatic activity is greater at pH 3 than at pH 5. Bacteria such as B. cenocepacia strain P525 may have value in the agricultural industry as biocontrol agents.     

<Emphasis Type="Italic">Syzygium aromaticum</Emphasis> extract mediated,rapid and facile biogenic synthesis of shape-controlled (3D) silver nanocubes

The synthesis of metal nano materials with controllable geometry has received extensive attention of researchers from the past decade. In this study, we report an unexplored new route for rapid and facile biogenic synthesis of silver nanocubes (AgNCs) by systematic reduction of silver ions with crude clove (Syzygium aromaticum) extract at room temperature. The formation and plasmonic properties of AgNCs were observed and the UV–vis spectra show characteristic absorption peak of AgNCs with broaden region at 430 nm along with the intense (124), (686), (454) and (235) peak in X-ray diffraction pattern confirmed the formation and crystallinity of AgNCs. The average size of AgNC cubes were found to be in the range of ~80 to 150 nm and it was confirmed by particles size distribution, scanning and transmission electron microscopy with elemental detection by EDAX. Further FTIR spectra provide the various functional groups present in the S. aromaticum extract which are supposed to be responsible and participating in the reaction for the synthesis of AgNCs. The AgNCs casted over glass substrate show an electrical conductivity of ~0.55 × 10⁶ S/m demonstrating AgNCs to be a potential next generation conducting material due to its high conductivity. This work provides a novel and effective approach to control the shape of silver nanomaterial for impending applications. The current synthesis mode is eco-friendly, low cost and promises different potential applications such as biosensing, nanoelectronics, etc.     

Methods for sequencing oligoribonucleotides and RNA by high performance liquid chromatography.

Conditions were established for the separation and quantitative determination of ribonucleosides, mono- and oligo-ribonucleotides by high-performance liquid chromatography (HPLC) on columns of AS-Pellionex SAX and AL-Pellionex WAX. By combining a high-speed UV spectrum monitor with an HPLC apparatus, products of RNase digestions of oligonucleotides and 5S ribosomal RNA (rRNA) were identified by measuring their UV spectra under continuous solvent flow, and also from their retention times on the columns (positions of elution). It took only 10 to 30 min for one chromatography run and required less than 0.01 A260 unit of sample per nucleotide material in each peak.     

Flavonoid glycosides and a bitter principle from lomatogonium carinthiacum

Three flavone glycosides, including the novel 6-hydroxyluteolin 7-O-gentiobioside, and a bitter secoiridoid glycoside, swertiamarin, have been identified in the aerial parts of Lomatogonium carinthiacum. Four minor xanthone glycosides were also detected. Separations were made with droplet counter-current chromatography (DCCC) in combination with column chromatography. Structure elucidation was achieved by UV, ¹H and ¹³C NMR, FDMS and D/CIMS spectra.     

Determination of huperzine A in formulated products by reversed-phase-liquid chromatography using diode array and electrospray ionization mass spectrometric detection.

A precise and selective high-performance liquid chromatographic (HPLC) method with diode-array detection for quantifying huperzine A in formulated products was developed and validated. A liquid chromatographic-mass spectrometric (LC/MS) procedure was devised to confirm the HPLC method. Huperzine A was dissolved in 1,2-dichloroethane, chromatographed on a YMCBasic C18 column, and detected at 308 nm. A gradient mobile phase of 10 mM ammonium acetate (pH = 3.5)--methanol was used. Identification was based on retention time, UV spectra and mass spectra by comparison with a commercial standard. The UV peak areas were used for quantitation of huperzine A content. The correlation coefficient (R2) of the calibration curve was 1 over the range 0.8-11.6 microg/ml. Overall recovery of huperzine A was 103.9% +/- 1.8 (mean +/- SD). Relative standard deviations for intra- and interday precision were < 2%.     

N5-(4-hydroxybenzyl) glutamine, 4-hydroxybenzylamine and 4-hydroxybenzylglucosinolate in Sinapis species

N⁵-(4-hydroxybenzyl) glutamine has been isolated from Sinapis alba L. and S. arvensis L. The identification is based on data obtained by HPLC, paper chromatography, high voltage electrophoresis, UV and NMR spectroscopy of the amide and its degradation products. The amide occurs together with 4-hydroxybenzylamine and sinalbin in both seeds and seedlings of the Sinapis species. This co-occurrence is briefly discussed in relation to chemotaxonomy, glucosinolate catabolism and amine metabolism.     

Identification of uric acid as the redox molecule secreted by the yeast Arxula adeninivorans

The yeast Arxula adeninivorans has been previously shown to secrete a large amount of an electro-active molecule. The molecule was produced by cells that had been cultivated in a rich medium, harvested, washed and then suspended in phosphate-buffered saline (PBS). The molecule was easily detectable after 60 min of incubation in PBS, and the cells continued to produce the molecule in these conditions for up to 3 days. The peak anodic potential of the oxidation peak was 0.42 V, and it was shown to be a solution species rather than a cell-attached species. We have optimised the production of the molecule, identified it by high-pressure liquid chromatography (HPLC) fractionation and high-resolution mass spectrometric analysis and determined the pathway involved in its synthesis. It has a mass/charge ratio that corresponds to uric acid, and this identification was supported by comparing UV spectra and cyclic voltammograms of the samples to those of uric acid. An A. adeninivorans xanthine oxidase gene disruption mutant failed to produce uric acid, which added further validity to this identification. It also demonstrated that the purine catabolism pathway is involved in its production. A transgenic A. adeninivorans strain with a switchable urate oxidase gene (AUOX) accumulated uric acid when the gene was switched off but did not when the gene was switched on. Cultivation of cells on amino acid and purine-free minimal media with an inorganic nitrogen source suggests that the cells synthesise purines from inorganic nitrogen and proceed to degrade them via the normal purine degradation pathway.     

Production of allantoin, rabdosiin and rosmarinic acid in callus cultures of the seacoastal plant Mertensia maritima (Boraginaceae)

A callus culture of the extreme halophyte seacoastal plant Mertensia maritima (Boraginaceae) was established from apical shoots of the plant using a modified Murashige and Skoog medium supplemented with 6-benzyladenine (0.5?mg/L) and ??-naphthaleneacetic acid (2.0?mg/L). Three main compounds, (?)-R-allantoin, (+)-rabdosiin and rosmarinic acid, were isolated from extracts of M. maritima calli by liquid chromatography and identified by ¹H and ¹³C NMR, UV, ECD and HPLC?CMS. Quantitative HPLC analysis showed that the calli produce (+)-rabdosiin (0.14% dry wt), rosmarinic acid (0.74% dry wt) and (?)-R-allantoin (3.7% dry wt). Allantoin was detected in plant cell cultures for the first time. All of these metabolites were also present in lower quantities in different parts of the plant. The presence of rabdosiin and rosmarinic acid, in combination with the skin-conditioning agent (?)-R-allantoin, represents a potentially useful novel composition for skin protection.     

Studies of the induction of mitotic gene conversion by ultraviolet irradiation: II. Action spectra

Action spectra for the induction of intragenic mitotic recombination (gene conversion) at the trp5 locus by UV are presented for three cell stages (T₀, T₉ and T₁₆) taken from synchronously growing cultures of Saccharomyces cerevisiae. The spectra over the range from 230 to 300 nm were taken mostly in 5-nm steps. The peak of action spectra was significantly shifted, regardless of the stage, toward the longer wavelengths as compared with that of the absorption spectrum of DNA (258 nm) or even that of thymine (265 nm). In one extreme case (T₁₆), the peak was shifted 17 nm from the absorption peak of DNA. Further, the spectrum changed its shape at the cell stage advanced from non-dividing (unbudded) (T₀) to a dividing phase (T₁₆). Furthermore, the induction cross section decreased by a large factor (about 40), regardless of the wavelength, in going from T₀ to T₁₆. From observations of the high photoreversibility of induced conversions, the major primary damage was thought to be pyrimidine dimers in the DNA.One plausible explanation, though not quite satisfactory from the quantitative viewpoint for these findings was that the increasing RNA during growth would screen the incident UV differentially with respect to the stage. If this explanation is correct, thymine dimers may still be considered, in spite of the shifts and deformations in the action spectra, as the major primary damage that triggers the long series of processes leading to gene conversion. Conventional methods for obtaining action spectra are discussed in comparison with the present method, which was based on sensitivity parameter a in the proposed dose (t)-frequency (f) relation, f = (at)^α (α is the multiplicity parameter).     

HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements

Methods for the detection and quantitation of hydroethidine (HE) and its oxidation products by HPLC analysis are described. Synthetic methods for preparation of authentic standards (2-hydroxyethidium and diethidium) are provided. Potential applications of the HPLC methods to chemical and biological systems are discussed. Specific examples of chromatograms obtained using UV–Vis absorption, fluorescence, electrochemical, and mass spectrometry detectors are provided. The development of a dual electrochemical and fluorescence detection methodology and its applications are described. The HPLC-based method enables analyses of HE and its oxidation products such as ethidium and the dimeric products of HE. The ramifications of HPLC measurement of HE and its oxidation products in the detection and quantitation of 2-hydroxyethidium, the diagnostic marker product of superoxide and HE, in the intracellular milieu are discussed. Similarly, mitochondria-targeted HE conjugated to a triphenylphosphonium group (Mito-HE or Mito-SOX) also forms oxidation products (dimers of Mito-HE and Mito-E⁺) that can affect the detection and quantitation of 2-hydroxy-mito-ethidium, the diagnostic marker product of Mito-HE and superoxide in mitochondria.     

Determination of aloesin in rat plasma using a column-switching high-performance liquid chromatographic assay

A column-switching high-performance liquid chromatography (HPLC) method for the determination of aloesin in rat plasma using column-switching and ultraviolet (UV) absorbance detection is described. Plasma was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The determination of aloesin was accurate and repeatable, with a limit of quantitation of 10 ng/ml in plasma. The standard calibration curve for aloesin was linear (r=0.998) over the concentration range of 10–1000 ng/ml in rat plasma. The intra- and inter-day assay variabilities of aloesin ranged from 1.0 to 4.7% and 1.1 to 8.8%, respectively. This highly sensitive and simple method has been successfully applied to a pharmacokinetic study after oral administration of aloesin to rats.     

2-O-(p-Coumaroyl)-l-Malate, 2-O-Caffeoyl-l-Malate and 2-O-Feruloyl-l-Malate in Raphanus sativus

The cotyledons, leaves and inflorescences of the radish, Raphanus sativus, have been examined for their content of hydroxylated cinnamic acid malate esters. 2-O-(p-coumaroyl)-l-malate, 2-O-caffeoyl-l-malate and 2-O-feruloyl-l-malate were found to be quantitatively predominating compounds in the fraction of carboxylic acids isolated from radish leaves and inflorescences. Identifications were based on PC, TLC, HVE, GC, HPLC, UV and NMR spectroscopy. The l- or (2S)-configuration of malic acid released by hydrolysis from the esters was determined by using l-malate dehydrogenase. A combined column chromatography technique applied prior to HVE, HPLC and GC was useful for separating the malate esters from most other low MW plant constituents. The significance of the present investigation is briefly discussed in relation to the metabolism of phenolic constituents of glucosinolate-containing plants, and the effects of these compounds in relation to insect feeding behaviour.     

Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421

Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as α and (β subunits by SDS-PAGE and MALDI-TOF. HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that α and β-subunits contained one and two phycocyanobilin groups as chromophores, respectively.     

Use of liquid chromatography to measure chlorite production by Nitrobacter winogradskyi NRCC26538

A high-pressure liquid chromatography (HPLC) technique, previously developed for nitrite (NO⁻₂) and nitrate (NO⁻₃) measurements [3], was used to measure chlorite (ClO⁻₂) production by Nitrobacter winogradskyi. The determination of ClO⁻₂ by HPLC involves monitoring the column effluent with a UV detector at 214 or 254 nm. Although the absorbance of ClO⁻₂ at 214 nm was about 5 times greater than at 254 nm, interference from other compounds in the culture filtrates of N. winogradskyi contributed to an unstable detector signal. The detection limit at 254 nm for ClO⁻₂ in deionized water was about 1 μM.The measurement of ClO⁻₂ in N. winogradskyi culture filtrates was done with detection at 254 nm. The maximum concentration of ClO⁻₂ produced by anaerobically incubated cell suspensions of N. winogradskyi was about 80 μM.