Comparison of protein precipitation methods for sample preparation prior to proteomic analysis

Protein samples should be free of salt and other disturbing agents and have an appropriate concentration to be suitable for two-dimensional (2D) electrophoresis, the principal step of proteomics. To find the most efficient method for sample preparation, we used human plasma and compared four widely applied precipitation methods, using trichloroacetic acid (TCA), acetone, chloroform/methanol and ammonium sulfate, as well as ultrafiltration. Precipitation with TCA and acetone and ultrafiltration resulted in an efficient sample concentration and desalting. We also found that ammonium sulfate fractionation can efficiently remove albumin, which represents more than 50% of plasma proteins.     

Statistical validation of sulfate quantification methods used for analysis of acid mine drainage

Turbidimetric method (TM), ion chromatography (IC) and inductively coupled plasma atomic emission spectrometry (ICP-AES) with and without acid digestion have been compared and validated for the determination of sulfate in mining wastewater. Analytical methods were chosen to compare the performance of a portable field turbidimetric instrument and to validate the underlying assumption utilized in conversion of total sulfur to sulfate during ICP-AES analysis. Accuracy and precision of analytical techniques were compared to one another using control and field samples collected from a mine site using the Bonferroni multiple comparison test. Effects of sample dilution, filter pore size and acidification on sulfate quantification were also studied. The results showed that IC and ICP-AES with and without acid digestion provided excellent recoveries in the case of control samples (within 90-110%). These analytical methods also showed lower relative standard deviation for both control and field samples. On the other hand, performance of the turbidimetric method was severely affected by sample dilution and acidification, and also revealed poor sulfate recoveries for control samples ranging from 0 to 83.5%. Analysis of variance (ANOVA) was used to evaluate the response (sulfate concentration) obtained from factorial design. Analytical method had significant effect (P < 0.0001) on the sulfate quantification. The interaction between determination method and sample dilution was more significant than other two-way interactions.     

Development and Validation of a Column-Switching LC–ESI–MS Assay for Determination of Huperzine A in Rat Plasma and Cerebrospinal Fluid

A simple and rapid method using liquid chromatography-mass spectrometry was developed and applied to determine the concentration of huperzine A in rat plasma or cerebrospinal fluid following a single intravenous injection or nasal administration. The chromatographic separation of the analytes was performed by column switching. A Zorbax SB-C₁₈ pre-column was used as the first column for sample clean-up, and then the analytes were eluted onto a Zorbax SB-C₁₈ column, the second column, and detected by electrospray ionization MS using single ion monitoring in positive mode. Hup B was used as internal standard. Linear calibration curve was achieved over a dynamic range of 0.5–500 ng mL^?1 for Hup A in plasma sample and 0.5–200 ng mL^?1 in CSF sample. The inter- and intra-assay coefficients of variation for the analysis were within ±6.67%. The mean absolute recoveries of Hup A from plasma were between 94.7 and 106.7%.     

Centrifugal precipitation chromatography,a powerful technique for the isolation of active enzymes from tea leaves (Camellia sinensis)

Centrifugal precipitation chromatography was developed approximately 10 years ago. In contrast to other counter-current chromatographic techniques, the centrifugal precipitation chromatography system is operated with two mutually miscible solutions separated by a cut-off membrane. Centrifugal precipitation chromatography was firstly introduced for the separation of proteins using an ammonium sulfate gradient. In this study we describe a novel approach using solvent-based protein precipitation for the isolation of active plant enzymes from tea leaves (Camellia sinensis) by centrifugal precipitation chromatography. We developed a gradient based on acetone and Tris-buffer, because the biological activity of carotenases in tea leaves cannot be preserved in the presence of ammonium sulfate. Parameters such as the critical solvent concentration, flow rate, buffer concentration, and sample load were determined and/or optimized. Subsequently, the newly developed separation protocol was successfully used for the isolation of active carotenoid cleavage enzymes from tea leaves. The isolated enzymes showed high enzymatic activities and purities and could be directly used for enzymatic assays and structure elucidation.     

Trace Enrichment on Pre-Columns in High Performance Liquid Chromatography. The Adsorption Capacity of a Reversed Phase Column

Abstract

The use of break-through experiments in investigations of the adsorption step in pre-column trace enrichment has been demonstrated.

The adsorption capacity of LiChrosorb RP-8 for phenol, 2,4-dichlorophenoxyacetic acid, ethylbenzene and pentachlorophenol has been determined as a function of the pH, ionic strength and flow-rate of the sample solution and as a function of the concentration of test substance in the sample.

The slope of the break-through curves has been found to show very little variation with the experimental conditions.     

Quantitative Determination of Fumonisins B1 and B2 by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

A high-performance liquid chromatographic method has been developed for the quantitative determination of the recently described mycotoxins, fumonisins B₁ (FB₁) and B₂ (FB₂) utilizing pre-column derivatization with o-phthaldialdehyde, isocratic elution, and fluorescence detection. Prior to analysis, sample extracts were purified on strong anion exchange cartridges. The method has been applied to the analysis of naturally contaminated corn and mixed horse feed samples as well as fungal culture material, for the presence of the mycotoxins. Detection limits are approximately 50 ng g^?1 for FB₁ and 100 ng g^?1 for FB₂. The method proved to be highly reproducible and recoveries of the toxins from the purification steps were found to be 99.5% and 85.9% for FB₁ and FB₂, respectively.     

Determination of total sulfite in wine. Zone electrophoresis-isotachophoresis quantitation of sulfate on a chip after an in-sample oxidation of total sulfite

This work deals with the determination of total sulfite in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP-ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite. At the same time, conductivity detection, employed to the detection of sulfate in the ZE stage of the ITP-ZE combination, provided for sulfate the concentration limit of detection corresponding to a 90 microg/l concentration of sulfite in the loaded sample (0.9 microl). Such a detectability allowed a reproducible quantitation of total sulfite when its concentration in wine was 15 mg/l. Formaldehyde binding of free sulfite in wine, included into the pre-column sample preparation, prevented an uncontrolled oxidation of this sulfite form. This step contributed to an unbiased determination of sulfate present in the original wine sample (this determination corrected for the concentration of sulfate determined in the sample after the peroxide oxidation of sulfite to the value equivalent to the total sulfite). The 99-101% recoveries of sulfite, determined for appropriately spiked wine samples, indicate a very good accuracy of the present method. Such a statement also supports excellent agreements of the results of quantitation based on the in-sample peroxide oxidation of the total sulfite (bound sulfite released at a high pH) with those in which this analyte was isolated from wine by distillation (bound sulfite released at a very low pH).     

Use of micellar mobile phases and microbore column switching for the assay of drugs in physiological fluids

The feasibility of directly assaying drugs in physiological fluids using on-line preconcentration and microbore high-performance liquid chromatography has been demonstrated. The untreated sample is injected onto a hydrophobic pre-column, using micellar sodium dodecyl sulfate (SDS) in the case of serum or phosphate buffer in the case of urine, as the load mobile phase. This traps the components of interest which are then backflushed onto a microbore analytical column using a stronger mobile phase. This procedure was then applied to diazepam in serum and phenobarbital in urine. Recovery was linear and quantitative over the range 30-3000 ng/ml for diazepam in serum and 2-200 micrograms/ml for phenobarbital in urine. The diazepam method was specific against caffeine and the three major metabolites of diazepam: oxazepam, temazepam, and nordiazepam. The effects of varying pre-column dimensions, pre-column loading time, and SDS concentration volume were evaluated.     

High Performance Liquid Chromatographic Assay of Hydromorphone Hydrochloride Injection

Abstract

A stability indicating high performance liquid chromatographic (HPLC) method for determining hydromorphone hydrochloride in dosage forms is described. The drug was chromatographed on a C₁₈ reverse phase column, using a mobile phase consisting of sodium lauryl sulfate, acetic acid, acetonitrile and water, and detected at 280 nm. Linearity of detector response to the concentration was confirmed. The procedure showed excellent reproducibility and gave quantitative recoveries of the drug from spiked placebos. Photodegraded samples of the dosage form, were assayed by the HPLC procedure and the current USP spectrophotometric procedure. Comparison of the results showed that the USP procedure is only partially stability indicating.     

Quantitative HPLC Analysis of Plasma Amino Acids as Orthophthaldialdehyde/Ethanethiol Derivatives

Abstract

A reverse phase high performance liquid chromatographic method for the analysis of plasma amino acids is described. The method employs pre-column derivatization with o-phthaldialdehyde using ethanethiol as the reducing agent. The analysis shows good linearity and reproducibility. An average overall difference of 12% was seen for results obtained by the HPLC method versus those obtained with an amino acid analyzer. The chromatographic parameters of buffer concentration and column temperature were also examined.     

Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic-coated capillaries

In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6 min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4 x 10? plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.     

The Effect of Silanol Masking on the Recovery of Picloram and Other Solutes from a Hydrocarbonaceous Pre-analysis Extraction Column

Abstract

The recoveries of picloram, picloram-methylester, hexazinone, benzene, and acetophenone from aqueous samples were studied using a commercially available hydrocarbonaceous pre-analysis extraction cartridge, both with and without tetrabutylammonium hydrogen sulfate (TBAHS) in the eluent. Extraction efficiency was found to be dependent on sample loading volume. The results suggest a mixed mechanism of retention involving both “silanophilic” and “hydrophobic” interactions in the absence of tetrabutylammonium ion. The ability of TBAHS to mask surface silanol groups and/or ion-pair with counterionic solutes is invoked to explain the observations. Chromatograms of the solutes obtained on a C₁₈ bonded analytical column in both the presence and absence of TBAHS are also presented.     

The Application of Trichloroacetic Acid as an Ion Pairing Reagent in LC–MS–MS Method Development for Highly Polar Aminoglycoside Compounds

A simple, sensitive and robust liquid chromatography-tandem mass spectrometry method was developed and validated for highly polar aminoglycoside compounds gentamicin, kanamycin and apramycin. The effect of trichloroacetic acid (TCA) concentration on plasma protein precipitation and sample recovery was studied and an optimized concentration of 25–30% TCA were determined that gives the best sample recovery for aminoglycosides from rat plasma. The effect of TCA concentration on the chromatographic behavior of gentamicin and tobramycin was studied on a Synergy Max RP-column using a mobile phase with a pH of 2.78. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 1–5,000, 20–10,000, 10–10,000 ng mL^?1 with precisions of 2.6–4.1, 3.3–5.0, 1.5–9.9%, and accuracies of 94.7–103.7, 87.9–104.9, 91.3–103.6% for gentamicin, kanamycin and apramycin, respectively. The LLOQs corresponding with a coefficient of variation less than 20% were 1, 20 and 10 ng mL^?1 for gentamicin, kanamycin and apramycin, respectively.     

Evaluation of an extraction method in the determination of the 2,4,6-trichloroanisole content of tainted cork

A method based on solvent extraction and GC-electron-capture detection analysis for the determination of 2,4,6-trichloroanisole (TCA) from cork has been evaluated and optimised. Our sample treatment consists of an extraction stage with pentane while the sample and solvent are kept in contact in a mechanical shaker (shake-flask extraction). Different extraction conditions have been tested in order to find the best compromise between efficiency and time of analysis. Different columns were evaluated for use in the concentration and purification step. A silica column was found to give the best performance in terms of recovery of TCA and repeatability. Pentane and mixtures of pentane-diethyl ether at different ratios were tested as eluting agents. It was found that 10 ml pentane allowed the recovery of retained TCA. Finally, the eluate was concentrated and injected into the chromatograph for TCA determination. The optimised chromatographic conditions enabled the quantification of TCA and 2,6-dichloroanisole, which was assayed as the internal standard. The shake-flask extraction method was compared with Soxhlet and ultrasound assisted extraction procedures using pentane as a solvent. Similar results were obtained for the shake-flask and Soxhlet extraction methods, while sonication gave significantly lower recoveries. The optimised shake-flask method was applied to determine the distribution of TCA in naturally contaminated cork bark.     

Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry

A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography–tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C₁₈ stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1–112.9 % and repeatability relative standard deviations of 1.9–7.2 %. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae.

Ionic liquid/Ammonium Sulfate Aqueous Two-phase System Coupled with HPLC Extraction of Sulfadimidine in Real Environmental Water Samples

Room temperature ionic liquids are novel solvents with a rather specific blend of physical and solution properties that make them of interest for applications in separation science. In this study, a green, simple, and sensitive sample pretreatment procedure coupled with high-performance liquid chromatography (HPLC) was developed for the analysis of sulfadimidine (SM2) that exploits an aqueous two-phase system based on 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF₄) and ammonium sulfate ((NH₄)₂SO₄) using a liquid?Cliquid extraction technique in real environmental water samples. The influences of the concentration of (NH₄)₂SO₄, pH value, temperature, and concentration of SM2 on the extraction efficiency of SM2 were determined. Under optimal conditions, the extraction efficiencies of SM2 were over 93.7%. Calibration curves yielded good linearity (R ² = 0.9998) over the range 3?C240 ng mL^?1, and the limit of detection and limit of quantification for analytes were 0.9 ng mL^?1 and 3 ng mL^?1, respectively. The proposed method was successfully applied to the quantification of SM2 in water samples and recoveries were in the range of 101.2?C107%.     

Application of factorial design to the study of xylitol production from eucalyptus hemicellulosic hydrolysate

This study deals with the bioconversion of xylose into xylitol by Candida guilliermondii FTI 20037 using eucalyptus hemicellulosic hydrolysate obtained by acid hydrolysis. The influence of various parameters (ammonium sulfate, rice bran, pH, and xylose concentration) on the production of xylitol was evaluated. The experiments were based on multivariate statistical concepts, with the application of factorial design techniques to identify the most important variables in the process. The levels of these variables were quantified by the response surface methodology, which permitted the establishment of a significant mathematical model with a coefficient determination of R ²=0.92. The best results (xylitol=10.0 g/L, yield factor=0.2 g/g, and productivity=0.1 g/[L·h]) were attained with hydrolysate containing ammonium sulfate (1.1 g/L), rice bran (5.0 g/L), and xylose (initial concentration of 60.0 g/L), after 72 h of fermentation. The pH of fermentation was adjusted to 8.0 and the inoculum level utilized was 3 g/L.     

Addition of urea and thiourea to electrophoresis sample buffer improves efficiency of protein extraction from TCA/acetone‐treated smooth muscle tissues for phos‐tag SDS‐PAGE

Phosphorylation analysis by using phos‐tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC₂₀) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)‐fixed tissues. Standard SDS sample buffer extracted less LC₂₀, actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4–5 fold. Phos‐tag SDS‐PAGE separated dephosphorylated and phosphorylated LC₂₀s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone.     

A Multicolumn Ion Chromatographic Determination of Nitrate and Sulfate in Waters Containing Humic Substances

Abstract

A three-column ion chromatographic system for the removal of humic substances from natural waters, and subsequent on-line concentration and determination of nitrate and sulfate using non-suppressed ion chromatography is presented. Humic substances are removed using disposable adsorption columns packed with chemically bonded amine silica material. The sample is directly transfered to an ion exchange column where the anions are concentrated ca 10 times. After reversing the flow, the ions are transferred to a third column where they are separated and quantified. The detection limit is less than 1 mg L^?1 of nitrate or sulfate in water containing 45mgL^?1 of humic acid.     

Single Cell Protein Production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Using an Optimized Culture Medium Composition in a Batch Submerged Bioprocess

Saccharomyces cerevisiae PTCC5269 growth was evaluated to specify an optimum culture medium to reach the highest protein production. Experiment design was conducted using a fraction of the full factorial methodology, and signal to noise ratio was used for results analysis. Maximum cell of 8.84 log (CFU/mL) was resulted using optimized culture composed of 0.3, 0.15, 1, and 50 g L^?1 of ammonium sulfate, iron sulfate, glycine, and glucose, respectively at 300 rpm and 35 °C. Glycine concentration (39.32 % contribution) and glucose concentration (36.15 % contribution) were determined as the most effective factors on the biomass production, while Saccharomyces cerevisiae growth had showed the least dependence on ammonium sulfate (5.2 % contribution) and iron sulfate (19.28 % contribution). The most interaction was diagnosed between ammonium sulfate and iron sulfate concentrations with interaction severity index of 50.71 %, while the less one recorded for glycine and glucose concentration was equal to 8.12 %. An acceptable consistency of 84.26 % was obtained between optimum theoretical cell numbers determined by software of 8.91 log (CFU/mL), and experimentally measured one at optimal condition confirms the suitability of the applied method. High protein content of 44.6 % using optimum culture suggests that Saccharomyces cerevisiae is a good commercial case for single cell protein production.