Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Selective determination of famotidine in human plasma by high performance liquid chromatography in alkaline media with solid phase extraction

A new method is described for the determination of famotidine by solid phase extraction from alkalinized human plasma followed by reversed phase (RP) HPLC in acetonitrile/alkaline buffer with molsidomine as an internal standard. Different acetonitrile/aqueous buffer mobile phases as well as various RP columns were used. Alkaline medium allowed the limit of quantitation to be lowered to 5 ng/mL of plasma as the famotidine gives more intense absorption at about 286 nm (at pH values higher than 7). Moreover, work in alkaline media and at this wavelength is highly selective as peaks corresponding to impurities present in most samples are well separated. A method using a mildly alkaline mobile phase (acetonitrile/10 mM phosphate with 10 mM 1‐heptanesulphonic acid, pH 7.5) was successfully used for determination of famotidine in human plasma in a pharmacokinetic study.     

Development and validation of a micellar electrokinetic capillary chromatography method for the determination of goserelin and related substances

An MEKC method for the analysis of goserelin and related substances has been developed using a combination of additives including CTAB, β‐CD, and sodium hexanesulfonate. For this assay, the running buffer (pH and additives) and separation conditions (voltage and temperature) were optimized. The optimized system was the following: 200 mM 6‐aminocaproic acid buffer (pH 4.2) supplemented with 175 mM CTAB, 3.0% w/v β‐CD, and 20 mM sodium hexanesulfonate; the voltage was 10 kV in reverse polarity mode, the temperature was 20°C, and UV detection was measured at 220 nm. The method was qualified by evaluating the specificity, precision, linearity, accuracy, LOD, and LOQ. According to validation experiments, the optimized method was specific, accurate, and repeatable and satisfied the requirements for the analysis of goserelin and related substances. Compared with the RP‐HPLC method, the MEKC method better solved the problem of overlapping impurity signals, and the migration time required was shorter. This method can be used for quality control and for the analysis of goserelin and its related substances.     

Simultaneous determination of cefoxitin,cefuroxime, cephalexin and cephaloridine in plasma using HPLC and a column-switching technique

Summary A new high performance liquid chromatographic method was developed using a column-switching technique for the simultaneous determination of cephalexin, cefuroxime, cefoxitin and cephaloridine in plasma. The plasma samples were injected onto a precolumn packed with Corasil RP C₁₈ (37–50 m) after simple dilution with an internal standard solution in 0.01 M acetate buffer (pH 3.5). Polar plasma components were washed out using 0.01 M acetate buffer (pH 3.5). After valve switching, the concentrated drugs were desorbed in back-flush mode and separated on a Partisil ODS-3 column using acetonitrile in 0.02 M acetate buffer (pH 4.3) (1585, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed with a detection limit of 0.5 g/ml. The total analysis time per sample was less than 25 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9 %.This method has been successfully applied to plasma from rats after subcutaneous injection of cefuroxime.     

Utilization of zirconia stationary phase as a tool in drug control

Zirconia-based stationary phases represent an interesting alternative to silica-based materials. Two zirconia-based stationary phases were studied as an option for use in drug analysis. The different properties of zirconia material, distinct from RP silica-columns, were employed for the development of a novel and rapid stability monitoring HPLC method. This method enables simultaneous control of possible degradation processes of active substance (ibuprofen) as well as antimicrobial excipients (methyl-and propylparaben). The separation of ibuprofen, its two main degradation products 2-(4-isobutyrylphenyl)propionic acid and 4-isobutylacetophenone, parabens, and 4-hydroxybenzoic acid as their degradation product was successfully accomplished on a Zr-CarbonC18 column using a mobile phase consisting of acetonitrile-phosphate buffer (pH 4.8)-propan-2-ol (27:56:17, v/v/v). Detection was performed at 258 nm and the analysis was completed within 17 minutes.     

Bioconversion of Kraft Paper Mill Sludges to Ethanol by SSF and SSCF

Paper mill sludge is a solid waste material composed of pulp residues and ash generated from pulping and paper making processes. The carbohydrate portion of the sludge has chemical and physical characteristics similar to pulp. Because of its high carbohydrate content and well-dispersed structure, the sludges can be biologically converted to value-added products without pretreatment. In this study, two different types of paper mill sludges, primary sludge and recycle sludge, were evaluated as a feedstock for bioconversion to ethanol. The sludges were first subjected to enzymatic conversion to sugars by commercial cellulase enzymes. The enzymatic conversion was inefficient because of interference by ash in the sludges with the enzymatic reaction. The main cause was that the pH level is dictated by CaCO₃ in ash, which is two units higher than the pH optimum of cellulase. To alleviate this problem, simultaneous saccharification and cofermentation (SSCF) using cellulase (Spezyme CP) and recombinant Escherichia coli (ATCC-55124), and simultaneous saccharification and fermentation (SSF) using cellulase and Saccharomyces cerevisiae (ATCC-200062) were applied to the sludges without any pretreatment. Ethanol yields of 75–81% of the theoretical maximum were obtained from the SSCF on the basis of total carbohydrates. The yield from the SSF was also found to be in the range of 74–80% on the basis of glucan. The SSCF and SSF proceeded under stable condition with the pH staying near 5.0, close to the optimum for cellulase. Decrease of pH occurred due to carbonic acid and other organic acids formed during fermentation. The ash was partially neutralized by the acids produced from the SSCF and SSF and acted as a buffer to stabilize the pH during fermentation. When the SSF and SSCF were operated in fed-batch mode, the ethanol concentration in the broth increased from 25.5 and 32.6 g/L (single feed) to 45 and 42 g/L, respectively. The ethanol concentration was limited by the tolerance of the microorganism in the case of SSCF. The ethanol yield in fed-batch operation decreased to 68% for SSCF and 70% for SSF. The high-solids condition in the bioreactor appears to create adverse effects on the cellulase reaction.     

Fast HPLC method for the determination of glimepiride, glibenclamide, and related substances using monolithic column and flow program

This work presents a fast method for the simultaneous separation and determination of glimepiride, glibenclamide, and two related substances by RP LC. The separation was performed on a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column. As mobile phase, a mixture of phosphate buffer pH 3, 7.4 mM, and ACN (55:45 v/v) was used. Column oven temperature was set to 30 degrees C. The total chromatographic run time was 80 s. This was achieved using a flow program from 5 to 9.9 mL/min. Precisions of the interday and the intraday assay for both retention times and peak areas for the four analyzed compounds were less than 1.2%. The method showed good linearity and recovery. The short analysis time makes the method very valuable for quality control and stability testing of drugs and their pharmaceutical preparations.     

Electrophoretron: a new method for enhancing resolution in electrokinetic separations

Until now no liquid chromatography (LC) method is described to determine the purity and content of troleandomycin and its related substances. A simple, robust, sensitive and selective isocratic liquid chromatographic method suitable for the determination of the antibiotic troleandomycin and its related substances is described. This method utilizes as a stationary phase: XTerra RP18 5 microm (25 cm x 4.6 mm I.D.) at 30 degrees C and as mobile phase: acetonitrile-0.2 M ammonium acetate buffer (pH 6.0)-water (45:5:50, v/v), delivered at a flow-rate of 1.0 ml/min. UV detection is performed at 205 nm. Troleandomycin is separated from the partially acetylated related substances and from several unknown impurities present in commercial samples. The robustness of the method was evaluated by a full-factorial experimental design.     

Determination of α-aminonitriles, α-amino acid amides and α-amino acids by means of HPLC,post-column reaction and fluorescence detection

Summary An HPLC method was developed for the simultaneous assay of intermediate (α-aminonitrile and α-amino acid amide) and end products (α-amino acid) in process streams of α-amino acid synthesis. Applications are given for Ala, Val and Leu. α-Aminonitriles were stable in a phosphate buffer pH 3, which was subsequently used for sample handling and chromatography. The α-aminonitrile, the corresponding acid amide and α-amino acid were separated using a buffered ion-pair mobile phase on an RP column and were detected fluorimetrically after post-column reaction with fluorescamine. Linearity and precision of the method are given.     

Simultaneous saccharification and fermentation of cellulosic biomass to acetic acid

Astrain of Clostridium thermoaceticum (ATCC 49707) was evaluated for its homoacetate potential. This thermophilic anaerobe best produces acetate from glucose at pH 6.0 and 59°C with a yield of 83% of theoretical. Enzyme hydrolysis of two substrates, a-cellulose and a pulp mill sludge, yielded 68% and 70% digestion, respectively. The optimum conditions for the simultaneous saccharification and fermentation (SSF) were substrate dependent: 55°C, pH 6.0 for α-cellulose, and 55°C, pH 5.5 for the pulp mill sludge. In the SSF with α-cellulose, the overall yield of acetate was strongly influenced by the enzyme loading. In a fed-batch operation of SSF with α-cellulose, an overall acetic acid yield of 60 wt% was obtained. Among the factors limiting the yields were incomplete digestion by the enzyme and the end-product inhibition. In the SSF of pulp mill sludge, inhibitors present in the sludge severely limited bacterial action. A large accumulation of glucose developed over the entire process, changing the intended SSF operation into a separate hydrolysis and fermentation operation. Despite a long lag phase of microbial growth, a terminal yield of 85% was obtained with this substrate.     

Design and Evaluation of an Optimal Controller for Simultaneous Saccharification and Fermentation Process

Ethanol from corn is produced using dry grind corn process in which simultaneous saccharification and fermentation (SSF) is one of the most critical unit operations. In this work an optimal controller based on a previously validated SSF model was developed by formulating the SSF process as a Bolza problem and using gradient descent methods. Validation experiments were performed to evaluate the performance of optimal controller under different process disturbances that are likely to occur in practice. Use of optimal control algorithm for the SSF process resulted in lower peak glucose concentration, similar ethanol yields (13.38±0.36% v/v and 13.50±0.15% v/v for optimally controlled and baseline experiments, respectively). Optimal controller improved final ethanol concentrations as compared to process without optimal controller under conditions of temperature (13.35±1.28 and 12.52±1.19% v/v for optimal and no optimal control, respectively) and pH disturbances (12.65±0.74 and 11.86±0.49% v/v for optimal and no optimal control, respectively). Cost savings due to lower enzyme usage and reduced cooling requirement were estimated to be up to $1 million for a 151 million L/yr (40 million gal/yr) dry grind plant.     

Analysis of Non-Selective Calcium-Channel Blockers by Reversed-Phase TLC

The selectivity of TLC separation of non-selective calcium-channel blockers prenylamine, lidoflazine, bepridil, and fendiline has been investigated silanized silica gel RP8 and RP18 plates. Optimization of retention and selectivity for these compounds was achieved by altering the pH and the concentration of organic modifier (methanol, ethanol, tetrahydrofuran, acetonitrile) in the aqueous mobile phases. The substances were separated in horizontal chambers and the drugs were detected by videoscanning and illumination of the plates at λ = 254 nm. On RP8 plates the best separation was achieved with 50% acetonitrile in pH 2.06 phosphate buffer as mobile phase. On RP18 the best separation was achieved with 50% ethanol in pH 2.06 phosphate buffer.

     

Development and validation of an LC method for the determination of emtricitabine and related compounds in the drug substance

A robust, precise, sensitive, linear, and simple RP LC method coupled with UV for the determination of emtricitabine or 2′,3′‐dideoxy‐5‐fluoro‐3′‐thiacytidine (FTC) and its related substances is described. The method uses an RP C18 column (25 cm×4.6 mm i.d.), 5 μm kept at a temperature of 35°C. The mobile phases for gradient elution consist of ACN, phosphate buffer (pH 4.4), and water. The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. A system suitability test (SST) was developed to verify the adequate performance of the chromatographic system. The developed method was further validated with respect to robustness, precision, sensitivity, and linearity. A central composite design was applied to examine the robustness of the method. The method shows good precision, sensitivity, linearity, and robustness. Three commercial FTC samples were examined using this method. This method is suitable to be used for the determination of related substances and assay of FTC.     

Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation

This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Simultaneous chiral discrimination of multiple profens by cyclodextrin-modified capillary electrophoresis in normal and reversed polarity modes

Simultaneous enantioseparations of nine profens for their accurate chiral discrimination were achieved by capillary electrophoresis (CE) in the normal polarity (NP) mode with a single cyclodextrin (CD) system and in the reversed polarity (RP) mode with a dual CD system. The single CD system in the NP mode employed heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) added at 75 mM-100 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) as the optimum run buffer. The dual CD system operated in the RP mode used 30 mM TMbetaCD and 1.0% anionic carboxymethyl-beta-cyclodextrin dissolved in pH 3.0, 100 mM phosphoric acid-triethanolamine buffer containing 0.01% hexadimethrine bromide added to reverse the electroosmotic flow. Fairly good enantiomeric resolutions and the opposite enantiomer migration orders were achieved in the two modes. Relative migration times to internal standard under respective optimum conditions were characteristic of each enantiomer with good precision (< 2% relative standard deviation, RSD), thereby enabling to crosscheck the chemical identification of profens and also their accurate chiralities. The method linearity in the two modes was found to be adequate (r > or = 0.9991) for the chiral assay of the profens investigated. Simultaneous enantiomeric purity test of ibuprofen, ketoprofen and flurbiprofen in a mixture was feasible in a single analysis by the present method.     

High-performance liquid chromatographic determination of some anthraquinone and naphthoquinone dyes occurring in historical textiles

A reversed-phase HPLC method has been developed for identification and quantitation of nine natural quinone dyes and applied to historical textile fibres. A Purospher RP18e column was used with a convex gradient of methanol in a mobile phase of 0.1 M aqueous citrate buffer (pH 2.5) and spectrophotometric diode-array detection at 270 nm. For identification of alizarin, purpurin and xanthopurpurin, occurring together in the madder plant, an isocratic method was used with a methanol-0.2 M acetate buffer (pH 4.3) (75:25) as the mobile phase. After an acid extraction of textile fibres and the analysis of the extracts, alizarin and purpurin were identified and quantitated in three fibres.     

Partial Characterization of Inulinases Obtained by Submerged and Solid-State Fermentation Using Agroindustrial Residues as Substrates: A Comparative Study

Inulinase belongs to an important class of enzymes as it can be used to produce high-fructose syrups by enzymatic hydrolysis of inulin and fructooligosaccharides, which has been used as functional food. This work aimed to carry out a partial characterization of the crude enzymatic extract of two different inulinases, obtained by solid-state fermentation (SSF) and submerged fermentation (SmF), using agroindustrial residues as substrates. The crude enzymatic extract obtained by SmF showed an optimal pH and temperature for hydrolytic activity of 4.5 and 55?°C, respectively; and that obtained by SSF conducted to optimal pH and temperature of 5.0 and 55?°C, respectively. Both enzymes presented high thermostability, with a D value of 230.4 h and 123.1 h for SmF and SSF, respectively. The inulinase produced by SmF showed highest stability at pH?4.4, while inulinase obtained by SSF was more stable at pH?4.8. The results showed that inulinase obtained by SmF is less susceptible to pH effect and the inulinase obtained by SSF is more resistant to higher temperatures.     

Determination of Doxorubicin,Daunorubicin and some of their Metabolites in Mouse Plasma by High-Performance Reversed-Phase Liquid Chromatography with Amperometric Detection

Abstract

A selective and sensitive reversed-phase liquid chromatographic method with electrochemical detection for the analysis of doxorubicin, daunorubicin and some of their metabolites in plasma is reported. A mobile phase consisting of acetonitrile-phosphate buffer solution-tetrahydrofuran (25–71,5–3,5) flowing at 1 ml/min through a Lichrocart RP 18 column was employed. The influence of various parameters on the separation (solvent composition, pH, tetrahydrofuran content) has been examined. An extraction of anthracyclines from plasma was performed using chloroform-ethanol mixture (4: 1) with high extraction efficiency; reproducible results were attained by working with a 1 M phosphate buffer which ensured a real buffering of the plasma samples. The sensitivity of amperometric detection makes this method suitable for analyzing small amounts of the parent drugs and their metabolites. The precision was better than 4% in the range 0.2 to 5 μg/ml plasma.     

High-performance liquid chromatographic determination of rifapentine in serum using column switching.

A high-performance liquid chromatographic method with column switching has been developed for the determination of rifapentine in serum. The serum samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard in a 1% ascorbic acid solution. Polar serum components were washed out using 0.05 M phosphate buffer. After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by a mu Bondapak C18 column with acetonitrile-tetrahydrofuran-0.05 M phosphate buffer (pH 7.0) (42:5:53, v/v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.1 microgram/ml. The total analysis time was less than 25 min and the mean coefficients of variation for intra- and inter-assay were less than 4.8%. The method has been successfully applied to serum samples from dogs after the oral administration of rifapentine.     

Electrochemical study of zolpidem at glassy carbon electrode and its determination in a tablet dosage form by differential pulse voltammetry

The oxidative behaviour of, a hypnotic drug, zolpidem was studied at glassy carbon electrode in Britton-Robinson buffer over the pH range 2.0-11.0 using cyclic, linear sweep and differential pulse voltammetry. Oxidation of the drug was effected in a single irreversible, diffusion-controlled step. Using differential pulse voltammetry (DPV), the drug yielded a well-defined voltammetric response in Britton-Robinson buffer, pH 8.0 at +0.889 V (vs. Ag/AgCl) on glassy carbon electrode. This process could be used to determine zolpidem concentrations in the range 5.0 x 10(-7) M to 1.0 x 10(-5) M with a detection limit of 2.0 x 10(-7) M. The method was applied, without any interference from the excipients, to the determination of the drug in a tablet dosage form.