Salt-induced phase separation can effectively remove the acetonitrile from the protein sample after the preparative RP-HPLC

Acetonitrile (ACN)–water system is one of the most commonly used mobile phases in practical reverse-phase high-performance liquid chromatography (RP-HPLC). However, a higher concentration of ACN (normally greater than 60% (v/v)) is required to elute the target protein from the RP-HPLC column in which, further steps to remove the ACN from the protein samples are demanded. It has been demonstrated that the phase separation occurring under the sub-zero temperature could easily remove the majority of ACN from the effluent of RP-HPLC. Recently, we found that the comparable phase separation could be achieved by adding a small amount of proper salts, such as K₂HPO₄ and KH₂PO₄, and the phase separation could take place effectively at 4 °C where the protein-purification processes were usually carried out. In addition, the pH value of the solution could be maintained properly by using potassium phosphate buffer (pH 7.0). With an optimized condition for this salt-induced phase separation, we demonstrated that greater than 60% of ACN could be easily removed; on the other hand, more than 90% of water-soluble protein could be successfully recovered within five hours.     

Three-dimensional protein map according to pI,hydrophobicity and molecular mass

A three-dimensional method has been developed to map the protein content of cells according to pI, M(w) and hydrophobicity. The separation of complex protein mixtures from cells is performed using isoelectric focusing (IEF) in the liquid phase in the first dimension, non-porous silica (NPS) RP-HPLC in the second dimension and on-line electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) detection in the third dimension. The experimentally determined pI, M(w) and hydrophobicity can then be used to produce a three-dimensional map of the protein expression of a cell, where now each protein can be tagged by three independent parameters. The ESI-TOF-MS provides an accurate M(w) for the intact protein while the hydrophobicity dimension results from the RP-HPLC component of the separation. The elution time, or percent acetonitrile at time of elution, of the protein is related to the hyrophobicity, which is an inherent property of the protein. 3D protein maps can thus be generated showing pI, M(w) and % acetonitrile at time of elution as well as pI, M(w) and hydrophobicity. The potential of the 3D plot for effective mapping of proteins from cells compared to current 2D methods is discussed.     

An improved method for tissue long-chain acyl-CoA extraction and analysis

We report an extensively modified method for the extraction, solid-phase purification, and HPLC analysis of long-chain acyl-CoAs from tissues. Tissue samples were homogenized in a glass homogenizer in KH2PO4 buffer (100 mM, pH 4.9) and again after the addition of 2-propanol. Acyl-CoAs were then extracted from the homogenate with acetonitrile (ACN). The acyl-CoAs in the extract were bound to an oligonucleotide purification column and eluted using 2-propanol. This eluent was concentrated and then loaded onto a C-18 column and eluted using a binary gradient system in which solvent A was KH2PO4 (75 mM, pH 4.9) and solvent B was ACN containing 600 mM glacial acetic acid. Initial flow rate was 0.5 or 0.25 ml/min depending upon the tissue used. The HPLC eluent was monitoring at 260 nm. Our modifications increased the recovery of the extraction procedure to 70-80%, depending upon tissue, with high reproducibility and significantly improved separation of the most common unsaturated and saturated acyl-CoAs. We also report, for the first time, the mass (nanomoles per gram wet weight) of the most common polyunsaturated acyl-CoAs in rat heart, kidney, and muscle tissues. The modifications and high recovery permit the use of tissue samples of less than 100 mg, making this method useful for the analysis of small tissue amounts associated with mice.     

Enantiomeric separation of prothioconazole and prothioconazole‐desthio on chiral stationary phases

Prothioconazole is a type of broad‐spectrum triazole thione fungicide developed by the Bayer Company. Prothioconazole‐desthio is the main metabolite of prothioconazole in the environment. In our study, enantiomeric separation of prothioconazole and prothioconazole‐desthio was performed on various chiral stationary phases (CSPs) by high‐performance liquid chromatography (HPLC). It was found that polysaccharide CSPs showed better ability than brushing CSPs in enantiomeric separation. The successful chiral separation of prothioconazole could be achieved on self‐made Chiralcel OD, commercialized Chiralcel OJ‐H and Lux Cellulose‐1. Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralcel OD, Chiralpak AY‐H, Chiralpak AZ‐H, and Lux Cellulose‐1 realized the baseline separation of prothioconazole‐desthio enantiomers. Simultaneous enantiomeric separation of prothioconazole and prothioconazole‐desthio was performed on Lux Cellulose‐1 using acetonitrile (ACN) and water as mobile phase. In most cases, low temperature favored the separation of two compounds. The influence of the mobile phase ratio or type was deeply discussed. We obtained larger Rs and longer analysis time with a smaller proportion of isopropanol (IPA) or ethanol and more water content at the same temperature. The ratio of ACN and water had influences on the outflow orders of prothioconazole‐desthio enantiomers. This work provides a new approach for chiral separation of prothioconazole and prothioconazole‐desthio with a discussion of chiral separation mechanism on different CSPs.     

Replacement of acetonitrile by ethanol as solvent in reversed phase chromatography of biomolecules

Acetonitrile, which is a by-product of acrylonitrile synthesis, is the commonly used solvent in ion-pair reversed phase chromatography. In consequence of the decreasing demand for acrylonitrile due to the financial crisis, a worldwide shortage of acetonitrile is observed. Therefore, the aim of this study was to establish ion-pair reversed phase chromatographic assays using alternative eluents for acetonitrile and to decrease costs incurred hereby. We compared the performance of ion-pair reversed phase chromatography using acetonitrile with the alternative eluents methanol, ethanol and n-propanol, using monolithic reversed phase C5 as well as C18 chromatography columns. We used triethylammonium acetate (TEAA) and tetrabutylammonium sulfate (TBA) as representative cationic ion-pair reagents and trifluoroacetic acid (TFA) as representative anionic ion-pair reagent. For covering a large field of applications, we fractionated representative low, middle and high-molecular weight biomolecules, in particular dinucleoside polyphosphates, peptides, proteins and tryptic digested human serum albumin. Whereas the chromatographic characteristics of both methanol and n-propanol were partly insufficient, ethanol was characterised equally or partly even better in the matter of elution strength and separation quality compared to the eluent water–acetonitrile. In conclusion, ethanol is an appropriate alternative for acetonitrile in ion-pair reversed phase chromatography of biomolecules.     

Enantiomeric separation of norgestrel by reversed phase high-performance liquid chromatography using eluents containing hydroxypropyl-beta-cyclodextrin in stereoselective skin permeation study

The chiral separation of norgestrel enantiomers using reversed-phase high-performance liquid chromatography (RP-HPLC) was studied with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral mobile phase additive. The effect of mobile phase composition, concentration of HP-beta-CD and column temperature on enantioselective separation were investigated. The quantification properties of the developed RP-HPLC method were examined. A baseline separation of norgestrel enantiomers was achieved on a Agilent ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 5.0, 20 mM) containing 25 mM HP-beta-CD (30:70, v/v) with a flow rate of 1.0 ml/min. The UV detector was set at 240 nm. Calibration curves were linear (n=8) in the range of 0.2-25 microg/ml, the limit of detection and quantitation were 0.10 and 0.20 microg/ml, respectively, for racemic norgestrel. The values of RSD of repeatability and intermediate precision for spiked sample were less than 4.8%. The method was successfully applied to the enantioselective determination of this drug in stereoselective skin permeation study.     

Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.

Isolation of the crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii was performed using 5,000 ground eye-stalks extracted in methanol-acetic acid-water (90:1:9). After the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, cyano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroacetic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. The bioassay of CHH during purification was done by injection of eluate fractions into eyestalk-ablated prawns and determination of the ability of the fractions to elevate glucose in the haemolymph. A complete amino acid sequence analysis was performed on one isoform of CHH (Mar-CHH-1), consisting of 71 residues. The sequence of Mar-CHH-1 shows considerable similarity (45-68%) to CHHs reported in other crustaceans. It is expected that there might be more than one isoform of CHH in M. rosenbergii.     

Development and validation of a sensitive liquid chromatography/mass spectrometry method for quantitation of flavopiridol in plasma enables accurate estimation of pharmacokinetic parameters with a clinically active dosing schedule

A high-performance liquid chromatographic assay with tandem mass spectrometric detection was developed and validated for quantitation of the broad spectrum kinase inhibitor, flavopiridol, in human plasma. Sample preparation conditions included liquid-liquid extraction in acetonitrile (ACN), drying, and reconstitution in 20/80 water/ACN. Flavopiridol and the internal standard (IS), genistein, were separated by reversed phase chromatography using a C-18 column and a gradient of water with 25 mM ammonium formate and ACN. Electrospray ionization and detection of flavopiridol and genistein were accomplished with single reaction monitoring of m/z 402.09>341.02 and 271.09>152.90, respectively in positive-ion mode [M+H](+) on a triple quadrupole mass spectrometer. Recovery was greater than 90% throughout the linear range of 3-1000 nM. Replicate sample analysis indicated within- and between-run accuracy and precision to be less than 13% throughout the linear range. This method has the lowest lower limit of quantitation (LLOQ) reported to date for flavopiridol, and it allows for more accurate determination of terminal phase concentrations and improved pharmacokinetic parameter estimation in patients receiving an active dosing schedule of flavopiridol.     

Fast protein separation by reversed-phase high-performance liquid chromatography on octadecylsilyl-bonded nonporous silica gel. II. Improvement in recovery of hydrophobic proteins.

Recovery of hydrophobic proteins from an RP-HPLC column was improved using a fast-separation RP-HPLC system operated at room temperature. Hydrophobic proteins such as ovalbumin could be adequately eluted from a nonporous octadecylsilyl (C18) spherical silica gel with a particle diameter of 20 microns using steep gradient elution with a 0.1% aqueous trifluoroacetic acid-acetonitrile system at a constant flow rate of 4 ml/min. Recoveries improved under fast separation since the protein sample suffered only a slight amount of irreversible denaturation on the hydrophobic surface of the stationary phase. The fast-separation system was also applied to the separation of larger proteins such as apo-ferritin (443 kDa) and thyroglobulin (669 kDa) as well as egg white proteins.     

Chiral separation of unmodified α-hydroxy acids by ligand exchange HPLC using chiral copper(II) complexes of (S)-phenylalaninamide as additives to the eluent

Copper(II) complexes of (S)-phenylalaninamide have been successfully used for the direct enantiomeric separation of unmodified (R,S)-α-hydroxy acids in reversed phase high-performance liquid chromatography (RP-HPLC). The effect of various parameters (pH, eluent polarity, selector concentration) on enantioselectivity is discussed. Evidence is provided that a mechanism of ligand exchange is actually occurring during the chromatographic separation. The method is very convenient and easy to use, and the chiral selector is commercially available and can be recovered at the end of the analysis. A conventional achiral RP-ODS-2 column is used and no pretreatment of the samples is required. This method allows the accurate determination of the enantiomeric excess of α-hydroxy acids in synthetic and biological samples. © 1995 Wiley-Liss, Inc.     

Use of a Novel Sub‐2 µm Silica Hydride Vancomycin Stationary Phase in Nano‐Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano‐liquid chromatography (nano‐LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns‐AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74–4.17 and 1.39–1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41–6.09 and α 1.28–2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. Chirality 27:767–772, 2015. © 2015 Wiley Periodicals, Inc.     

Separation of recombinant β-glucuronidase from transgenic tobacco by aqueous two-phase extraction

Transgenic plants hold many promises as viable production hosts for therapeutic recombinant proteins. Many efforts have been devoted to increase the expression level of the proteins, but the efforts for developing economic processes to purify those proteins are lacking. In this report, aqueous two-phase extraction (ATPE) was investigated as an alternative for the separation of an acidic recombinant protein, β-glucuronidase (rGUS), from transgenic tobacco. Screening experiments by fractional factorial designs showed that PEG concentration and ionic strength of the system significantly affected the partitioning of native tobacco proteins and GUS. Response surface methodology was used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4% (w/w) PEG 3400/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while more than 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process. The most important impurity from tobacco, Rubisco, was largely removed from the rGUS in the recovered phase.     

Antibacterial activities in various tissues of the horse mussel, Modiolus modiolus

A search for antibacterial activity in different organs/tissues of the horse mussel, Modiolus modiolus, was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid. Due to high salt content, two liquid phases were obtained; an acetonitrile-rich phase (ACN extract) and an aqueous phase. The aqueous phase was further subjected to solid phase extraction (SPE). Eluates from SPE and ACN extracts were tested for antibacterial, lysozyme, and toxic activity. Antibacterial activity was demonstrated in extracts from several tissues, including plasma, haemocytes, labial palps, byssus, mantle, and gills. Some of the extracts were sensitive to proteinase K treatment, indicating antibacterial peptides and/or proteins. Lysozyme-like activity and toxic activity against Artemia salina nauplii was detected in fractions from the gills, mantle, muscle, and haemocytes. Results from this study indicate that M. modiolus is a promising source for identifying novel drug lead compounds.     

Investigation of the pharmacokinetics and determination of cholesteryl carbonate zidovudine in rat plasma by non-aqueous reversed-phase high performance liquid chromatography with UV detection

A simple reversed phase high-performance liquid chromatographic method was established for the separation and quantification of a novel prodrug of zidovudine in rat plasma. Zidovudine was one of the original drugs used to treat HIV infection. Appropriate aliquots of rat plasma were spiked with cholesteryl carbonate zidovudine (AZTC) and treated with acetonitrile to precipitate plasma proteins. The supernatant after supercentrifugation was collected and an aliquot of 20 microL was injected directly into an HPLC system consisting of a Diamonsil C18 column and a diode array detector. The mobile phase consisted of methanol (85%, v/v) and diethyl ether (15%, v/v) at a flow rate of 1.0 mL/min. The extraction recoveries of AZTC at the three concentrations examined were all higher than 80%. The HPLC assay was linear over the concentration range 0.5-80 microg/mL. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration-time profile for AZTC after administration via the tail vein. The mean elimination half-life (t1/2) was 292.4 min. This RP-HPLC method will be useful for the evaluation of the pharmacokinetics of AZTC in rats.     

Fluorescence and circular dichroism spectroscopy of cytochrome c in alkylammonium formate ionic liquids

The structural stability of cytochrome c has been studied in alkylammonium formate (AAF) ionic liquids such as methylammonium formate (MAF) and ethylammonium formate (EAF) by fluorescence and circular dichroism (CD) spectroscopy. At room temperature, the native structure of cytochrome c is maintained in relatively high ionic liquid concentrations (50-70% AAF/water or AAF/phosphate buffer pH 7.0) in contrast with denaturation of cytochrome c in similar solutions of methanol or acetonitrile with water or buffer cosolvents. Fluorescence and CD spectra indicate that the conformation of cytochrome c is maintained in 20% AAF-80% water from 30 to 50 °C. No such temperature stability is found in 80% AAF-20% water. About one-third of the enzyme activity of cytochrome c in 80% AAF-20% water can be maintained as compared with phosphate buffer, and this is greater than the activities measured in corresponding methanol and acetonitrile aqueous solutions. This biophysical study shows that AAFs have potential application as organic solvent replacements at moderate temperature in the mobile phase for the separation of proteins in their native form by reversed phase liquid chromatography.     

Cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phases as effective tools for enantioselective HPLC separation of structurally different disubstituted binaphthyls

Cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phases (CSPs) were used for a study of the HPLC retention and enantioseparation behavior of 2,2'-disubstituted or 3,2,2'-trisubstituted 1,1'-binaphthyls and 8,3'-disubstituted 1,2'-binaphthyls. The effects of the mobile phase composition in normal- (NP) and reversed-phase (RP) separation modes were investigated. The NP mobile phases contained n-hexane and propane-2-ol at various volume ratios, the RP ones were obtained by mixing acetonitrile with water or a 20 mM phosphate buffer of pH 6.0 or 3.0. The RP separation mode has been found more suitable for enantioresolution of most of the analytes. The best enantioseparation of 2,2'-diacetyl-1,1'-binaphthyl, 2-hydroxy-2'-(phenylamino)-1,1'-binaphthyl-3-carboxylic acid and 2-amino-2'-hydroxy-1,1'-binaphthyl-3-carboxylic acid was obtained in the mobile phase of ACN/20 mM phosphate buffer, pH 3.0, 40/60 (v/v), whereas N-(2'-hydroxy-1,1'-binaphthyl-2-yl)acetamide, N-(3'-methoxy-1,2'-binaphthyl-8-yl)acetamide, and N-(3'-hydroxy-1,2'-binaphthyl-8-yl)acetamide yielded better results in ACN/water at the same v/v ratio. The analyte-CSP interaction mechanism was found to be temperature independent but the enantioresolution improved at an elevated temperature. The mechanism of the enantioselective discrimination is discussed on the basis of the thermodynamic parameters obtained. Semi-preparative separation conditions have been proposed for 2-amino-2'-hydroxy-1,1'-binaphthyl-3-carboxylic acid, N-(3'-methoxy-1,2'-binaphthyl-8-yl)acetamide, and N-(3'-hydroxy-1,2'-binaphthyl-8-yl)acetamide.     

A systematical analysis of tryptic peptide identification with reverse phase liquid chromatography and electrospray ion trap mass spectrometry

In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identified peptides in reverse phase liquid chromatography and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Following protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identified peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol.Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state ( 2, 3) peptides are likely to be identified, and the hydropathicity of the peptides might not be related to whether it is more likely to be identified or not. The number of identified peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identified peptides present three types of redundancy, namely identification, charge, and sequence redundancy, which may repress low abundance protein identification.     

Screening for antibacterial and antifungal activities in marine benthic invertebrates from northern Norway

Benthic marine invertebrates collected from sub-Arctic regions of northern Norway, were found to be a promising source of novel bioactive compounds against human and fish pathogenic bacteria and fungi. Lyophilized material from seven species of ascidians, six sponges and one soft alcyonid coral were extracted with 60% acidified acetonitrile (ACN). After separation into an ACN-rich phase (ACN-extract) and an aqueous phase, and subsequent solid-phase extraction of the aqueous phase, fractions differing in polarity were obtained and screened for antibacterial and antifungal activities, along with the more lipophilic ACN-extracts. Antimicrobial activity was determined against two Gram-negative, two Gram-positive bacteria, and two strains of fungi. Notably, all the invertebrate species in the study showed activity against all four strains of bacteria and the two strains of fungi. In general, the aqueous fractions displayed highest antimicrobial activity, and the most potent extracts were obtained from the colonial ascidian Synoicum pulmonaria which displayed activity against bacteria and fungi at a concentration of 0.02 mg/ml; the lowest concentration tested.     

Biodegradation of acetonitrile by adapted biofilm in a membrane-aerated biofilm reactor

A membrane-aerated biofilm reactor (MABR) was developed to degrade acetonitrile (ACN) in aqueous solutions. The reactor was seeded with an adapted activated sludge consortium as the inoculum and operated under step increases in ACN loading rate through increasing ACN concentrations in the influent. Initially, the MABR started at a moderate selection pressure, with a hydraulic retention time of 16 h, a recirculation rate of 8 cm/s and a starting ACN concentration of 250 mg/l to boost the growth of the biofilm mass on the membrane and to avoid its loss by hydraulic washout. The step increase in the influent ACN concentration was implemented once ACN concentration in the effluent showed almost complete removal in each stage. The specific ACN degradation rate achieved the highest at the loading rate of 101.1 mg ACN/g-VSS h (VSS, volatile suspended solids) and then declined with the further increases in the influent ACN concentration, attributed to the substrate inhibition effect. The adapted membrane-aerated biofilm was capable of completely removing ACN at the removal capacity of up to 21.1 g ACN/m² day, and generated negligible amount of suspended sludge in the effluent. Batch incubation experiments also demonstrated that the ACN-degrading biofilm can degrade other organonitriles, such as acrylonitrile and benzonitrile as well. Denaturing gradient gel electrophoresis studies showed that the ACN-degrading biofilms contained a stable microbial population with a low diversity of sequence of community 16S rRNA gene fragments. Specific oxygen utilization rates were found to increase with the increases in the biofilm thickness, suggesting that the biofilm formation process can enhance the metabolic degradation efficiency towards ACN in the MABR. The study contributes to a better understanding in microbial adaptation in a MABR for biodegradation of ACN. It also highlights the potential benefits in using MABRs for biodegradation of organonitrile contaminants in industrial wastewater.