Lysozyme extraction from hen egg white in an aqueous two-phase system composed of ethylene oxide–propylene oxide thermoseparating copolymer and potassium phosphate

The recovery and purification of lysozyme from hen egg white has been investigated in an aqueous two-phase systems composed of thermoseparating random copolymers of ethylene oxide (EO), propylene oxide (PO) and potassium phosphate. In the primary extraction step lysozyme was satisfactorily partitioned to the top polymer-rich phase in a system composed of 40% (w/w) EO₅₀PO₅₀, 10% (w/w) potassium phosphate, and 0.85 M sodium chloride at pH 9.0, diluted 3-fold with crude egg white, where contaminating proteins were discarded in the bottom phosphate-rich phase. After the primary phase separation the upper EO₅₀PO₅₀ phase was removed and subjected to temperature-induced (65 °C) phase separation, which resulted in the partitioning of pure lysozyme to the top water phase. The separation system was found to be efficient in achieving the purification of lysozyme in a high yield of 85% and specific activity of 32,300 U/mg of protein, with a purification factor of 16.9 and a concentration of lysozyme in the water phase of 2.3 g/l in two extraction steps.     

Downstream processing of luciferase from fireflies (Photinus pyralis) using aqueous two-phase extraction

The firefly luciferase has been extensively used for sensitive detection of bacteria, gene expression and environmental toxins (biosensors). The aim of the present study was to design a simple and more efficient method for the purification and concentration of luciferase using aqueous two-phase extraction (ATPE). Downstream processing of luciferase from North American Firefly Photinus pyralis was carried out, for the first time, using polymer/salt aqueous two phase system (ATPS) at 4 °C. The enzyme was observed to preferentially partition to the polyethylene glycol (PEG) rich top phase. The best results of purification (13.69 fold) and enzyme activity recovery (118.34%) were observed in the system containing 4.0% (w/w) PEG (1500) and 20.5% (w/w) (NH₄)₂SO₄ with a phase volume ratio of 0.21.     

Separation and purification of laccases from two different fungi using aqueous two-phase extraction

Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)–phosphate aqueous two-phase system at 25 °C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and 0.013 g g⁻¹ of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.     

Extraction of peroxidase from bitter gourd (Momordica charantia) by three phase partitioning with dimethyl carbonate (DMC) as organic phase

Three phase partitioning (TPP) is most renowned technique used for extraction and purification of natural products. In previous studies of TPP, t-butanol is mainly used as an organic phase. This is the first report that explores ability of dimethyl carbonate (DMC) in the field of TPP as an alternate solvent for t-butanol. In the present study TPP process with t-butanol and DMC as organic phase along with different salts was applied to waste bitter gourd powder to obtained peroxidase enzyme. DMC was found to be compatible with most of salts such as ammonium sulphate and sodium citrate and explored as more efficient solvent than t-butanol. This TPP system provides 4.84 fold purity of peroxidase enzyme at optimum source concentration of 0.15 g/mL, with a system comprising DMC as organic phase, sodium citrate (20%) as salt, agitation speed 120 rpm, pH 7, temperature 30 °C and extraction time of 3 h. Present study has aimed for extraction and separation of peroxidase from bitter gourd waste with TPP technique and ensures the scope of carbonated solvents in extraction and purification of proteins.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

Determination of Tenacissoside A in rat plasma by liquid chromatography–tandem mass spectrometry method and its application to pharmacokinetic study

A sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for the first time for the estimation of Tenacissoside A in the rats’ plasma, which is the major active constituent in Marsdenia tenacissima. Tenacissoside A was extracted from the rats’ plasma by using liquid–liquid extraction (LLE), medroxyprogesterone acetate was used as the internal standard. An Alltech C18 column (250 mm × 4.6 mm, 5 μm) was used to provide chromatographic separation by detection with mass spectrometry operating in selected ion monitoring (SIM) mode. The method was validated over the concentration range of 1–250 ng/mL for Tenacissoside A. The precisions within and between-batch (CV%) were both less than 15% and accuracy ranged from 90 to 102%. The lower limit of quantification was 1 ng/mL and extraction recovery was 88.3% on average. The validated method was used to study the pharmacokinetic profile of Tenacissoside A in rat after administration.     

New HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib,dasatinib, and nilotinib in human plasma

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia.     

Optimized method for the determination of itopride in human plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.     

Extraction and purification of laccase by employing a novel rhamnolipid reversed micellar system

Biosurfactant-based reversed micellar extraction (RME) is an innovative method for separation and purification of biomolecules. In this study, rhamnolipid (RL), a kind of biosurfactant, was firstly adopted to form a novel reversed micellar system for extracting and purifying laccase from Coriolus versicolor crude extract. Several significant factors affecting both forward and backward extraction processes were studied. The appropriate conditions for forward extraction process were: 3.3 mM RL, 50 mM KCl, pH 5.5 and extracting time 40 min. As regards backward extraction process, 250 mM KCl, pH 7.0 and extracting time 40 min were suggested. The corresponding activity recovery (AR) and purification fold (PF) were 92.7% and 4.79, respectively. Electrophoresis analysis indicated that the laccase was successfully purified. After this reversed micellar system was reused three times, the AR and PF declined to 70.8% and 4.35, respectively, indicating that the reversed micellar system could be reused. Comparisons results of synthetic surfactant-based RME and RL-based RME further verified the superiority of RL.     

5CN05 partitioning in an aqueous two-phase system: A new approach to the solubilization of hydrophobic drugs

Liquid–liquid extraction for the purification of molecules has been central to many advances in the pharmaceutical industry. These processes were developed based on the property that some polymer and/or micellar solutions present to separate into a concentrated phase and a diluted phase. Based on the differences in the physical and chemical environments of the two coexisting phases, and since both phases contain approximately 60–90% of water, liquid–liquid extraction provides a powerful alternative to both extract and solubilize a molecule. This paper examines the partition behavior of the synthetic drug, 2-[(3,4-dichlorine-benzylidene)-amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (5CN05), in an aqueous two-phase polymer system (ATPPS) and also in an aqueous two-phase micellar system (ATPMS). The results showed that both systems are favorable for extraction the 5CN05 drug high partition coefficient values (K_5CN05 > 200) and yield (Y_5CN05 > 99.48%) in the concentrated phase were achieved with the systems. However, the ATPPS generated a partition coefficient (K_5CN05) higher than the one obtained with ATPMS. The results suggest that both processes may be used for the extraction and concentration of molecules with hydrophobic characteristics, such as 5CN05. They also provide an optimal environment for the solubilization of such molecules, allowing for greater efficiency when purifying many classes of drugs.     

Partial purification of extracellular exo-inulinase from Ulocladium atrum

Eight fungal species were cultivated on the Czapek liquid medium and a good starting extracellular and intracellular exo-inulinase were selected. Extracellular inulinase from Ulocladium atrum was prepared in the presence of 1% inulin source and 0.2% sodium nitrate as the best carbon and nitrogen sources. Incubation for the U. atrum was increased till it reached its maximum (36 U/ml) at the sixth day of incubation at 30 °C which was the best temperature for the production of exo-inulinase. Effect of all metal ions inhibited inulase production by U. atrum. Exo-inulinase was purified by using ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose. Three active inulinase forms INI, INII and INIII were resolved, each for DEAE cellulose. The specific activity of INI was 1915 U/mg protein which represented 2.65-fold purification over the crude extract with 42.8% recovery pooling of INI placed on CM cellulose chromatography and INI was resolved into INIa, INIb and INIc. The specific activity of INIa was 2479.2 U/mg protein which represented 3.43-fold purification over the crude extract with 24.2% recovery.     

Two-stage recovery of S-adenosylmethionine using supported liquid membranes with strip dispersion

We report the selective recovery of S-adenosylmethionine (SAM) from fermentation broths using a two-stage supported liquid membrane system with strip dispersion (SLM-SD). The system utilized two MiniModule^® hollow-fiber membrane modules as microporous supports and an organic membrane solution consisting of the extractants of sodium di-2-ethylhexyl sulfosuccinate (AOT), di-(2-ethylhexyl)phosphoric acid (DEHPA), and trioctylphosphine oxide (TOPO) in the solvent n-octanol. SAM was extracted in the first membrane module. Methionine (Met) was captured by the first stripping solution and further purified in the second membrane module. pH values in the feed phase and the first and second stripping solutions, extractant concentrations, NaCl concentration, and the SAM acceptor in the first stripping solution were optimized. Strip dispersion mixing speed, pressure differences across the membranes, and flow rates of the feed and strip dispersion phases were investigated experimentally. The optimal extractant concentrations were: AOT 2.78 wt%, DEHPA 27.0 wt%, and TOPO 1.61 wt%. The optimal pH values in the feed phase and the first and second stripping solution were 3.0, 2.5, and 1.0, respectively. SAM extraction efficiency of 98.7%, SAM recovery efficiency of 91.8% and Met removal efficiency of 85.4% were achieved within 5 h. Finally, the mass transfer analysis indicated that the mass transfer resistances from the extraction reaction and the membrane phase were predominant.     

Continuous countercurrent salting-out extraction of 1,3-propanediol from fermentation broth in a packed column

The possibility of continuous extraction of 1,3-propanediol in a experimental packed column was investigated using a salting-out extraction system of dipotassium phosphate/ethanol. Mass transfer of 1,3-propanediol takes place from the dispersed phase (salt-rich solution) to the continuous phase (ethanol). The influences of flow rate of dispersed phase and size of packing material on partition coefficient and recovery of 1,3-propanediol were investigated and the results were compared with those obtained in spray column and test tube. Furthermore, the influences of various system compositions on hold up of dispersed phase, mass transfer coefficient, and system stability were also studied in the column packed by stainless steel Dixon 3 × 3 mm. It was found that the packed column showed a good extraction efficiency and stability. Besides, 1,3-propanediol recovery of 90.30% was obtained during a 11 h continuous operation when the real fermentation broth was used. At the same time, 94.4% of phosphate could be recovered when 0.2 volume of anhydrous ethanol was added into the raffinate phase at pH 4.0.     

Single-step purification of recombinant green fluorescent protein on expanded beds of immobilized metal affinity chromatography media

Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (q_max) of Ni²⁺ Streamline™ and Ni²⁺ cross-linked alginate for the GFP was 1,42,860 FU ml⁻¹ and 18,000 FU ml⁻¹, respectively. The expanded bed column chromatography using Ni²⁺ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni²⁺ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.     

Development and validation of a high throughput and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of diltiazem and its two metabolites in human plasma: Application to a bioequivalence study

A high throughput and specific method using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of diltiazem and its two metabolite (N-desmethyldiltiazem and O-desacetyldiltiazem) in human plasma. A one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) involved for the extraction of diltiazem (DLTZ), metabolites (DMeD and DAcD) and internal standard. Analytes were chromatographed on a ACQUITY UPLC? BEH C₁₈ column (100 mm × 2.1 mm, i.d., 1.7 μm) with isocratic elution at a flow rate of 0.2 mL/min using 10 mM ammonium acetate buffer–acetonitrile (25:75, v/v). The Quattro Premier XE LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Using 300 μL plasma, the method was validated over the concentration range 0.48–639.9 ng/mL for DLTZ and 0.24–320.1 for DMeD and 0.24–320.7 ng/mL for DAcD, with a lower limit of quantification of 0.48 ng/mL for DLTZ and 0.24 ng/mL for metabolites. The intra- and inter-day precision and accuracy were within 10.0%. The recovery was 77.4%, 76.0%, 74.5% and 74.1% for DLTZ, DMeD, DAcD and Ziprasidone, respectively. Total run time was 2.0 min only.     

Speciation of organotin compounds in urine by GC–MIP-AED and GC–MS after ethylation and liquid–liquid extraction

A method for the determination of organotin compounds in urine samples based on liquid–liquid extraction (LLE) in hexane and gas chromatographic separation was developed and optimized. Seven organotin species, namely monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), tetrabutyltin (TeBT), monophenyltin (MPhT), diphenyltin (DPhT) and triphenyltin (TPhT), were in situ derivatized by sodium tetraethylborate (NaBEt₄) to form ethylated less polar derivatives directly in the urine matrix. The critical parameters which have a significant effect on the yield of the successive liquid–liquid extraction procedure were examined, by using standard solutions of tetrabutyltin in hexane. The method was optimized for use in direct analysis of undiluted human urine samples and ways to overcome practical problems such as foam formation during extraction, due to various constituents of urine are discussed. After thorough optimization of the extraction procedure, all examined species could be determined after 3 min of simultaneous derivatization and extraction at room temperature and 5 min phase separation by centrifugation. Gas chromatography with a microwave-induced plasma atomic emission detector (MIP-AED) as element specific detector was employed for quantitative measurements, while a quadrupole mass spectrometric detector (MS) was used as molecular specific detector. The detection limits were between 0.42 and 0.67 μg L^?1 (as Sn) for the quantitative LLE–GC–MIP-AED method and the precision between 4.2% and 11.7%, respectively.     

Refolding and purification of recombinant human granulocyte colony-stimulating factor using hydrophobic interaction chromatography at a large scale

High level expression of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in Escherichia coli (E. coli) usually forms insoluble and inactive aggregates, i.e. inclusion bodies. In the present work, high performance hydrophobic interaction chromatography (HPHIC) was applied to the refolding of rhG-CSF, which was solubilized by 8.0 mol L^?1 urea from the inclusion bodies. First a laboratorial scale column (10 mm × 20 mm I.D.) was employed to study the refolding process. Several factors, including concentration of ammonium sulfate, pH of the mobile phase and flow rate, were investigated in details. The results indicated that the rhG-CSF produced by E. coli could be successfully refolded with simultaneous purification by using HPHIC. The refolding process was further scaled up by using a large column (50 mm × 200 mm I.D.). 200 mL of rhG-CSF solution solubilized by 8.0 mol L^?1 urea, with a total amount of protein around 1.6 g, could be loaded onto the large column at one time. Under these conditions, the obtained rhG-CSF had a specific activity of 2.3 × 10⁸ IU mg^?1 and a purity of 95.4%, the mass recovery during the purification was 36.9%. This work might have great impact on practical production of rhG-CSF, and it also shed a light on protein refolding using liquid chromatography at large scales.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate aminoflavone (NSC 686288) in human plasma

A reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for determination of aminoflavone (AF) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 mL of plasma with 1.0 mL ethyl acetate containing 50 ng/mL of the internal standard zileuton. The analytes were separated on a Waters X-Terra? MS C₁₈ column using a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the AF concentration range of 5–2000 ng/mL in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL for AF in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully applied to characterize AF plasma concentration-time profile in the cancer patients in a phase I trial.     

Development of a sensitive and selective LC-MS/MS method for the determination of α-fluoro-β-alanine, 5-fluorouracil and capecitabine in human plasma

A sensitive and selective quantitative method to determine α-fluoro-β-alanine (FBAL), 5-fluorouracil (5-FU), and capecitabine (Cape) from a single human plasma aliquot (50 μL) has been developed and validated. First, 5-FU and Cape were extracted by liquid–liquid extraction (LLE) using a mixture of acetonitrile and ethyl acetate. This was followed by derivatization with dansyl chloride. The dansyl-derivatives from 5-FU and Cape were further purified using LLE with methyl tertiary-butyl ether (MTBE) and analyzed using a reversed-phase analytical column “Primesep D” (2.1 mm × 50 mm; 5 μm) with embedded basic ion-pairing groups. The remaining aqueous phase containing FBAL was treated with dansyl chloride and the dansyl-FBAL was purified by solid phase extraction. Ultra high pressure liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for analysis of dansyl-FBAL. The method was validated over the concentration ranges of 10–10,000, 5–5000, and 1–1000 ng/mL for FBAL, 5-FU, and Cape, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.