Ammonium Acetate Concentration Research Articles

Nonaqueous capillary electrophoresis for analysis of the ethanol consumption biomarker phosphatidylethanol

Nonaqueous CE (NACE) methodology was developed for the separation and determination of phosphatidylethanol (Peth), a new biomarker of ethanol intake. Peth is an abnormal phospholipid formed in cell membranes only in the presence of ethanol, via the transphosphatidylation reaction of phospholipase D. The NACE separation medium consisted of 80 mM ammonium acetate in 50% ACN, 33% 2-propanol, 12% hexane and 5% methanol. A stacking effect was obtained by reducing the concentration of ammonium acetate in the separation medium for all injected samples. The LOD was estimated to 1 microM (5.6 fmol) of Peth with conventional UV detection, equalling 0.4 micromol/L blood. Peth was successfully determined in extracts of human blood samples. Separation of Peth from other blood lipids in the lipid extract sample was performed in 5 min. The method facilitates smaller sample volumes and performs about ten times faster compared to earlier chromatographical methods.

Noncovalent Complexes Between DNA and Basic Polypeptides or Polyamines by MALDI-TOF

MALDI-MS was evaluated as a method for the study of noncovalent complexes involving DNA oligonucleotides and various polybasic compounds (basic polypeptides and polyamines). Complexes involving single-stranded DNA were successfully detected using DHAP matrix in the presence of an ammonium salt. Control experiments confirmed that the interactions involved basic sites of the polybasic compounds and that the complexes were not formed in the gas phase but were pre-existing in the matrix crystals. Moreover, the pre-existence in solution was probed by isothermal titration calorimetry at concentration and ionic strength similar to those used for mass spectrometry. Spectra showed no important difference between negative and positive ion modes. The influence of nature and size of DNA and polybasic compound on the relative intensities and stoichiometries of the complexes was investigated. Despite the fact that relative intensities can be affected by ionization yields and the gas-phase stabilities of the different species, numerous trends observed in the MALDI study were consistent with the expected in-solution behaviors. Experimental conditions related to sample preparation were investigated also. Complex abundance generally decreased when increasing the ammonium acetate concentration. It was dramatically decreased when using ATT instead of DHAP. Penta-L-arginine is an exception to these observations. Lastly, in the case of complexes involving DNA duplex, the ATT matrix was shown to favor the observation of specific DNA duplex but not that of its complex with polybasic compounds. Inversely, DHAP was appropriate for the conservation of DNA-polybasic compound interaction but not for the transfer of intact duplex.

Stability of [6]-gingerol and [6]-shogaol in simulated gastric and intestinal fluids

The degradation kinetics of [6]-gingerol and [6]-shogaol were investigated in simulated gastric (pH 1) and intestinal (pH 7.4) fluids at 37 °C. Degradation products were quantitatively determined by HPLC (Lichrospher 60 RP select B column, 5 μm, 125 mm × 4 mm; mobile phase: methanol–water–acetic acid (60:39:1 v/v); flow rate: 0.6 ml/min; detection UV: 280 nm). In simulated gastric fluid (SGF) [6]-gingerol and [6]-shogaol underwent first-order reversible dehydration and hydration reactions to form [6]-shogaol and [6]-gingerol, respectively. The degradation was catalyzed by hydrogen ions and reached equilibrium at approximately 200 h. In simulated intestinal fluid (SIF) both [6]-gingerol and [6]-shogaol showed insignificant interconversion between one another. Addition of amino acids glycine, 3-amino propionic acid (β-alanine) and γ-amino butyric acid (GABA), and ammonium acetate at a range of concentrations of 0.05–0.5 mM had no effect on the rate of degradation of [6]-shogaol in SGF and 0.1 M HCl solution. However, at exceedingly high concentration (0.5 M) of ammonium acetate and glycine, significant amounts of [6]-shogaol ammonia and glycine adducts were detected. The degradation profile of [6]-gingerol and [6]-shogaol under simulated physiological conditions reported in this study will provide insight into the stability of these compounds when administered orally.

Remaining acetamide in acetonitrile degradation using nitrile hydratase- and amidase-producing microorganisms

The tandem conversion process involving nitrile hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetonitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetonitrile hydration step catalyzed by nitrile hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetonitrile conversion process.

Determination of ximelagatran, melagatran and two intermediary metabolites in plasma by mixed-mode solid phase extraction and LC–MS/MS

An analytical method was developed for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and the two intermediate metabolites, OH-melagatran and ethyl-melagatran in human plasma. Extraction of plasma was carried out on a mixed mode bonded sorbent material (C8/SO 3 −). All four analytes, including their isotope-labelled internal standards, were eluted at high ionic strength with a mixture of 50% methanol and 50% buffer (0.25 M ammonium acetate and 0.05 M formic acid, pH 5.3) with an extraction recovery above 80%. The extracts were demonstrated to be clean in terms of a low concentration of albumin and lysoPC. The sample extraction was fully automated and performed in 96-well plates using a Tecan Genesis pipetting robot. Analysis of the extracts were performed with liquid chromatography followed by positive electrospray ionization mass spectrometry. The low organic content and the low pH of the extracts allowed for, after dilution 1:3 with buffer, direct injection onto the LC-column. The four analytes were separated on a C18 analytical LC-column using gradient elution with the acetonitrile concentration varying from 10 to 30% (v/v) and the ammonium acetate and acetic acid concentration kept constant at 10 and 5 mmol/L, respectively, at a flow rate of 0.75 mL/min. Linearity was achieved over the calibrated range 0.010–4.0 μmol/L with accuracy and relative standard deviation in the range 96.9–101.2% and 6.6–17.1%, respectively at LLOQ, and in the range 94.7–102.6% and 2.7–6.8%, respectively at concentrations above 3 × LLOQ. The method replaces a manual method, and displays the advantages of having a fully automated sample clean-up, no evaporation/reconstitution step, high recovery, and complete LC-separation of all four analytes.

Synthesis of hydrated CeO2 nanowires and nanoneedles

Hydrated CeO2 nanowires as thin as 5 nm in diameter and nanoneedles with various aspect ratios were obtained via a chemical precipitation technique in the presence of ammonium acetate. It was possible to control the particle size, shape and agglomeration by tuning the ammonium acetate concentration in the experimental solutions. The effect of ammonium acetate on the particle morphological parameters is discussed and a possible mechanism is suggested.

Behaviour of sulphonated azodyes in ion-pairing reversed-phase high-performance liquid chromatography

Ion-pairing reversed-phase high-performance liquid chromatography (RP-HPLC) operation conditions were studied to obtain information useful for development and optimization of separation methods suitable for HPLC/MS analysis of dyes. The retention of eight sulphonated azodyes with widely differing structures (1–5 acid groups, molecular weight range 350–1220) was measured in mobile phases containing various ion-pairing reagents in aqueous-methanolic mobile phases. The effects of the type and of the concentration of ammonium acetate, tetrabutylammonium hydrogen sulphate and five di- and tri-alkylammonium acetate ion-pairing reagents on the chromatographic behaviour of dyes were compared in mobile phases with varying concentrations of methanol. Structural effects on the retention of dyes were studied in detail. The retention scale based on lipophilic and polar indices can be used for optimization of mobile phase for HPLC/MS of dyes and, on the other hand, may provide some information on the structure of unknown dyes.

HPLC Separation of Digested Proteins and Preparation for Matrix-Assisted Laser Desorption/Ionization Analysis

INTRODUCTIONTwo types of columns are commonly used for the separation of peptides by HPLC. A single-phase column contains the reverse-phase resin C18, which interacts with the hydrophobic moieties of the peptides. Peptides resulting from digestion of simple mixtures of proteins are loaded onto the single-phase column and eluted into the mass analyzer using an increasing gradient of an organic solvent. Peptides resulting from the digestion of more complex mixtures of proteins are resolved using a biphasic column. This column integrates both a strong cation exchange SCX resin, which interacts with peptides as a result of their positive charge, and a reverse-phase C18 resin, packed in tandem. Peptides initially interact with the SCX resin and are eluted into the C18 resin by ammonium acetate that competes for the peptide-binding sites. Peptides are then eluted from the C18 resin into the mass analyzer. This process is repeated using increasing concentrations of ammonium acetate to differentially elute peptides in a stepwise fashion. The biphasic column also uses an additional C18 reverse-phase resin linked through an Inline MicroFilter Assembly (Upchurch) to desalt the peptides prior to loading onto the SCX. The desalting and biphasic columns are combined to give an integrated desalting/biphasic column.

Biochemical, Molecular, and Functional Characterization of PISCF-Allatostatin, a Regulator of Juvenile Hormone Biosynthesis in the Mosquito Aedes aegypti

Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10(-9) m. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti.

Capillary electrochromatography‐mass spectrometry of cationic surfactants

The CEC-MS of alkyltrimethylammonium (ATMA+) ions with chain lengths ranging from C1-C18 is optimized using an internally tapered column packed with mixed mode reversed phase/strong cation exchange stationary phase. A systematic study of the CEC separation parameters is conducted followed by evaluation of the ESI-MS sheath liquid and spray chamber settings. First, the optimization of CEC separation parameters are performed including the ACN concentration, triethylamine (TEA) content, buffer pH and ammonium acetate concentration. Using 90% v/v ACN with 0.04% v/v TEA as mobile phase, the separation of longer chain C6-C18-TMA+ surfactants could be achieved in 15 min. Lowering the ACN concentration to 70% v/v provided resolution of shorter chain C1, C2-TMA+ from C6-TMA+ although the total analysis time increased to 40 min. Furthermore, variation of both the ACN and TEA content as well as ionic strength has found to significantly influence the retention of longer chain surfactants as compared to shorter chains. The optimum CEC conditions are 70% v/v ACN, 0.04% v/v TEA, pH 3.0 and 15 mM ammonium acetate. Next, the optimization of the ESI-MS sheath liquid composition is conducted comparing methanol to isopropanol followed by the use of experimental design for analysis of spray chamber parameters. Overall, the developed CEC-ESI-MS method allows quantitative and sensitive monitoring of ATMA+ from < or =10 microg/mL down to 10 ng/mL. Utilizing the optimized CEC-ESI-MS protocol, the challenging analysis of commercial sample Arquad S-50 ATMA+ containing cis-trans unsaturated and saturated soyabean fatty acid derivatives is demonstrated.

Expression, purification and characterization of porcine pancreatic Carboxypeptidase B from Pichia pastoris for the conversion of recombinant human insulin

Pancreatic or tissue Carboxypeptidase B (CPB), a key enzyme involved in insulin conversion and highly specific for excising C-terminal Lys and Arg residues from peptides and proteins, was expressed at high level and purified from a recombinant Pichia pastoris strain. A cDNA containing the porcine pancreatic pro-Carboxypeptidase B (proCPB) fused to the Saccharomyces cerevisiae alpha factor secretion signal was cloned into the pPIC3K vector under control of P. pastoris AOX1 promoter. After 72 h of growth on methanol, proCPB accumulated until 320 mg L −1, representing 70% of total proteins in culture supernatant. A single stepwise ion exchange purification process with Q-Sepharose at increasing concentrations of ammonium acetate allowed recovery of 65% proCPB in a single fraction. The dialyzed protein was activated with trypsin and its activity was tested with the synthetic substrate Hippuryl- l-Arg. The kinetic parameters K M and V max, as well as inhibition constant K i against a specific inhibitor were calculated and found similar to those of the wild-type enzyme. The enzyme efficiently removed amino acids Lys and Arg from the spacer of an insulin precursor (B 1–30-LysArg-A 1–21) expressed in yeast and previously cleaved with trypsin. The enzyme was found stable for 4 h at pH 11.8, a useful property for performing both enzyme reactions with trypsin and CPB in a single step.

Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues

The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4 M) were compared with a phenol–chloroform extraction method and with a commercial DNA isolation kit. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR. The 167 and 268 bp fragments of APC and β-globin genes, respectively, were amplified equally from DNA extracted by all tested methods and in all cases. However, the 536 bp fragment of β-globin gene was not amplified in all cases. According to our results, the extraction method using ammonium acetate proved to be simple and suitable for obtaining DNA of good quality, which can be easily amplified by PCR.

Application of polymeric surfactants in micellar electrokinetic chromatography‐electrospray ionization mass spectrometry of benzodiazepines and benzoxazocine chiral drugs

Chiral micellar EKC (CMEKC) coupled to ESI-MS using polymeric surfactants as pseudostationary phases is investigated for simultaneous enantioseparation of two benzodiazepines, (+/-)-oxazepam ((+/-)-OXA) and (+/-)-lorazepam ((+/-)-LOR), and one benzoxazocine, (+/-)-nefopam ((+/-)-NEF). First, enantioselectivity and electrospray sensitivity of six chiral polymeric surfactants for all three chiral compounds are compared. Second, using poly(sodium N-undecenoyl-L-leucinate) as pseudostationary phase, the organic modifiers (methanol (MeOH), isopropanol, and ACN) are added into the running buffer to further improve chiral resolution (RS). Next, a CMEKC-ESI-MS method for the simultaneous enantioseparation of two benzodiazepines is further developed by using a dipeptide polymeric surfactant, poly(sodium N-undecenoxy carbonyl-L,L-leucyl-valinate) (poly-L,L-SUCLV). The CMEKC conditions including nebulizer pressure, capillary length, ammonium acetate concentration, pH, poly-L,L-SUCLV concentration, and capillary temperature were optimized to achieve maximum chiral RS and highest sensitivity of MS detection. The spray chamber parameters (drying gas temperature and drying gas flow rate) as well as sheath liquid conditions (MeOH content, pH, flow rate, and ionic strength) were found to significantly influence MS S/N of both (+/-)-OXA and (+/-)-LOR. Finally, a comparative study between simultaneous UV and MS detection showed high plate numbers, better chiral RS, and enhanced detectability with CMEKC-MS. However, speed of analysis was faster using CMEKC-UV.

Role of the Carbohydrate Binding Site of the Streptococcus pneumoniae Capsular Polysaccharide Type 3 Synthase in the Transition from Oligosaccharide to Polysaccharide Synthesis

The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)-beta-D-GlcUA-(1, 4)-beta-D-Glc-(1-]n. Synthesis is comprised of two distinct catalytic phases separated by a transition step whereby an oligosaccharylphosphatidylglycerol primer becomes tightly bound to the carbohydrate acceptor recognition site of the synthase. Using the recombinant synthase in Escherichia coli membranes, we determined that a critical oligosaccharide length of approximately 8 monosaccharides was required for recognition of the growing chain by the synthase. Upon binding of the oligosaccharide-lipid to the carbohydrate recognition site, the polymerization reaction entered a highly processive phase to produce polymer of high molecular weight. The initial oligosaccharide-synthetic phase also appeared to be processive, the duration of which was enhanced by the concentration of UDP-GlcUA and diminished by an increase in temperature. The overall reaction approached a steady state equilibrium between the polymer- and oligosaccharide-forming phases that was shifted toward the former by higher UDP-GlcUA levels or lower temperatures and toward the latter by lower concentrations of UDP-GlcUA or higher temperatures. The transition step between the two enzymatic phases demonstrated cooperative kinetics, which is predicted to reflect a possible reorientation of the oligosaccharide-lipid in conjunction with the formation of a tight binding complex.

Separation and determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation by nonaqueous capillary electrophoresis

An easy, rapid method for simultaneous determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation was developed by nonaqueous capillary electrophoresis (NACE) without pretreatment for the first time. Optimum separation was achieved with a fused-silica capillary column (50 cm × 75 μm i.d.) and a running buffer containing 30 mM ammonium acetate, 1.0% acetic acid and 15% acetonitrile (ACN) in methanol medium. The applied voltage was 30.0 kV. The analytes were detected by UV at 214 nm. The effects of concentration of ammonium acetate, acetic acid and organic modifier on electrophoretic behavior of the analytes were studied. The established method with sophoridine as internal standard was linear in the range of 5–1000 mg/mL for both strychnine and brucine. The extracts of Strychnos nux-vomica and its preparation could be directly injected for determination with recoveries ranging from 94.5 to 104%.

Hydrothermal synthesis and luminescent properties of YBO 3:Tb 3+ uniform ultrafine phosphor

The pure hexagonal phase YBO 3:Tb 3+ phosphors with good crystallinity and uniform size were prepared by hydrothermal reaction (HR). The particle size and morphology can be well controlled by adjusting the concentration of ammonium acetate and varying the reaction temperature and time. The phosphor prepared by HR emits an intense green light at 543 nm, which is stronger than that from crystals synthesized by solid-state reaction (SR). The influence of Tb 3+-doping concentration on the crystallization and luminescent properties were investigated. The results showed that the samples exhibited a higher quenching concentration of Tb 3+ in comparison with those prepared by the SR. The phenomena were also discussed.

The Turnover Kinetics of Major Histocompatibility Complex Peptides of Human Cancer Cells

Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins. Thus, the repertoire of these peptides (the MHC peptidome) should correlate better with the cellular protein degradation scheme (the degradome) than with the cellular proteome. To test the validity of this statement and to determine whether the majority of MHC peptides are derived from short lived proteins, from defective ribosome products, or from regular long lived cellular proteins we analyzed in parallel the turnover kinetics of both MHC peptides and cellular proteins in the same cancer cells. The analysis was performed by pulse-chase experiments based on stable isotope labeling in tissue culture followed by capillary chromatography and tandem mass spectrometry. Indeed only a limited correlation was observed between the proteome and the MHC peptidome observed in the same cells. Moreover a detailed analysis of the turnover kinetics of the MHC peptides helped to assign their origin to normal, to short lived or long lived proteins, or to the defective ribosome products. Furthermore the analysis of the MHC peptides turnover kinetics helped to direct attention to abnormalities in the degradation schemes of their source proteins. These observations can be extended to search for cancer-related abnormalities in protein degradation, including those that lead to loss of tumor suppressors and cell cycle regulatory proteins.

Rational method development strategies on a fluorinated liquid chromatography stationary phase: Mobile phase ion concentration and temperature effects on the separation of ephedrine alkaloids

Fluorinated, silica-based stationary phases are becoming increasingly popular alternatives to traditional alkyl phases owing to their differential selectivity and retention for a variety of analyte classes. In this report, the ion-exchange mechanisms characteristic of a fluorinated phase are exploited to rapidly develop separation conditions for ephedrine alkaloids and synephrine using a mobile phase compatible with mass spectrometry. A linear relationship of basic analyte retention with the reciprocal of ammonium acetate concentration is first established. This linear relationship can then be used to optimize retention and selectivity in just two experiments. The relationship of retention with temperature is also explored. Greater retention with increasing temperature is demonstrated on the fluorinated phase at high percentages of organic modifier, which is in contrast to behavior observed in typical reversed-phase separations. The unexpected observation is explicated based on the reduction in solvent solvating power with increasing temperature. As solvation power of the mobile phase decreases, decreased solvation of both mobile phase and ionized surface groups of the stationary phase leads to stronger interactions between analyte and stationary phase. Both mobile phase ion concentration and temperature are shown to be powerful tools for the manipulation of analyte retention and selectivity.

Thermal, electrical and optical studies on the poly(vinyl alcohol) based polymer electrolytes

Solid polymer proton conductors comprising of poly(vinyl alcohol), ammonium acetate and water have been prepared by solution cast method for different NH 4 +/OH − ratios. The XRD spectra for the electrolyte indicate that the amorphous nature of PVA increases with the concentration of ammonium acetate. The DSC curves show the low glass transition temperature for the ratio (NH 4 +/OH −) = 0.25 which relates to higher conductivity of the sample. The ionic conductivity at room temperature depends strongly on NH 4 +/OH − ratios. The variation of electrical conductivity with temperature showed two regions of activation above and below glass transition temperature. The optical absorption studies show the similar trend for pure PVA and salt-doped PVA with different absorption intensity. The direct and indirect band gap energy is observed to be constant for pure PVA and salt-doped PVA samples and found to be 5.4 eV and 4.8 eV, respectively. The dc polarization measurement shows that the conductivity is mainly due to ions.

Development of an assay method for the detection and quantification of protease and non-nucleoside reverse transcriptase inhibitors in plasma and in peripherical blood mononuclear cells by liquid chromatography coupled with ultraviolet or tandem mass spectrometry detection

We present a simple chromatographic method to detect and quantify protease inhibitors (PI), metabolites and non-nucleoside reverse transcriptase inhibitors (NNRTIs) in human plasma of HIV-1 infected patients and in peripheral blood mononuclear cells (PBMCs) using either liquid chromatography coupled with ultraviolet (LC–UV) or liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). A solid–liquid extraction was carried out on 500 μl of plasma as pre-treatment. Calibration curve ranges were from 50 (100) to 5000 ng/ml (indinavir). PBMC pellets from 7 ml of blood were lysed with methanol/tris with a calibration curve ranging from 0.25 to 250 ng/pellet. Simple modifications in the mobile phase composition (slight increase of ammonium acetate concentration and addition of methanol for LC–UV) easily linked the two analytical systems.