A Validated Chiral LC Method for the Enantiomeric Separation of Nateglinide and the Quantitative Determination of Its l-Enantiomer

A simple and accurate chiral liquid chromatographic method was developed for the enantiomeric purity determination of d-nateglinide and quantitative determination of l-nateglinide in bulk drug samples. Good resolution (R _s  > 6.0) between d-enantiomer and l-enantiomer of nateglinide were achieved with Chiralpak AD-H (250 × 4.6 mm, 5 μm particle size) column using hexane and ethanol (90:10 v/v) as mobile phase at 25 °C temperature. Flow rate was kept as 1.0 mL min^?1 and elution was monitored at 210 nm. The effects of the mobile phase composition, the flow rate and the temperature on the chromatographic separation were investigated. Developed method is capable to detect (LOD) and quantitate (LOQ) l-nateglinide to the levels of 0.3 and 1.0 μg mL^?1 respectively, for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of l-nateglinide at LOQ concentration was 5.2. The percentage recoveries of l-nateglinide from d-nateglinide ranged from 97.9 to 99.7. The test solution and mobile phase was found to be stable up to 24 h after preparation. The developed method was validated with respect to LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.     

Enantioselective Separation and Determination of Carnosine in Rat Plasma by Fluorescence LC for Stereoselective Pharmacokinetic Studies

A sensitive fluorescence liquid chromatographic analytical method was developed for the simultaneous determination of carnosine enantiomers in rat plasma. The method was applied to pharmacokinetic studies. Chiral separation of carnosine enantiomers was achieved by pre-column derivatization with o-phthaldialdehyde and the thiol N-acety-l-cysteine as derivating reagents. They were separated on an ODS column and detected by fluorescence detection (λ_ex = 350 nm, λ_em = 450 nm). γ-Aminobutyric acid was used as internal standard. The method was linear up to 6,000 ng mL^?1 for l-carnosine, 4,000 ng mL^?1 for d-carnosine. Low limit of quantitation (LLOQ) was 40 ng mL^?1 for each isomer. The relative standard deviations obtained for intra- and inter-day precision were lower than 12% and the recoveries were higher than 75% for both enantiomers. The method was applied to a stereoselective study on the pharmacokinetics of carnosine after oral administration with a single dose (carnosine, 75 mg kg^?1 for each isomer) to a rat. The initial data indicated that l-carnosine had a larger value of the highest plasma concentration than d-carnosine (C _max 5,344 vs. 1,914 ng mL^?1), and that of l-carnosine had a lower value of AUC_(0?∞) and t _1/2(h) (AUC_(0?∞) 5,306 vs. 6,321 ng h mL^?1, t _1/2 1.43 vs. 3.37 h). Our results indicated that the pharmacokinetic of l-carnosine and d-carnosine revealed enantioselective properties significantly.     

l-Ribose Production from l-Arabinose by Immobilized Recombinant Escherichia coli Co-expressing the l-Arabinose Isomerase and Mannose-6-Phosphate Isomerase Genes from Geobacillus thermodenitrificans

l-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the l-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l l-ribose from 300 g/l l-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co²⁺, with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h^?1 produced an average of 100 g/l l-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for l-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from l-arabinose as the substrate.     

Exploiting thermodynamic data to optimize the enantioseparation of underivatized amino acids in ligand exchange capillary electrophoresis

Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral separation of underivatized d,l-His by ligand exchange capillary electrophoresis (LECE), utilizing accurate ex ante calculations. This has been obtained by the addition to the background electrolytes (BGE) of NaClO₄ which renders the separations “all in solution processes”, allowing to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances. To this aim, the formation of ternary complexes of Cu²⁺ ion and l-lysine (l-Lys) or l-ornithine (l-Orn) with l- and d-histidine (His), and histamine (Hm) have been studied by potentiometry and calorimetry at 25 °C and with 0.1 mol dm^?3 (KNO₃) in aqueous solution. The ternary species [Cu(L)(l-His)H]⁺ and [Cu(L)(d-His)H]⁺ (where L?=?l-Lys or l-Orn) show a slight but still detectable stereoselectivity, and the determination of ΔH° and ΔS° values allowed the understanding of the factors which determine this phenomenon. The stereoselectivity showed by the protonated ternary species has been exploited to chirally separate d,l-His in LECE, by using the binary complexes of copper(II) with l-Lys or l-Orn as background electrolytes added with the appropriate amounts of NaClO₄.

Chiral resolution of l- and d-alanine and a racemic macrocyclic nickel(II) complex: synthesis and crystal structures

The reactions of a racemic four-coordinate Ni(II) complex [Ni(rac-L)](ClO₄)₂ with l- and d-alanine in acetonitrile/water gave two six-coordinate enantiomers formulated as [Ni(RR-L)(l-Ala)](ClO₄)·2CH₃CN (1) and [Ni(SS-L)(d-Ala)](ClO4) (2) (L = 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclo-tetradecane, Ala^? = alanine anion), respectively. Evaporation from the remaining solutions gave two four-coordinate enantiomers characterized as [Ni(SS-L)](ClO₄)₂ (S-3) and [Ni(RR-L)](ClO₄)₂ (R-3), respectively. Single-crystal X-ray diffraction analyses of complexes 1 and 2 revealed that the Ni(II) atom has a distorted octahedral coordination geometry, being coordinated by four nitrogen atoms of L in a folded configuration, plus one carboxylate oxygen atom and one nitrogen atom of l- or d-Ala^? in mutually cis-positions. Complexes 1 and 2 are supramolecular stereoisomers, constructed via hydrogen bonding between [Ni(RR-L)(l-Ala)]⁺ or [Ni(SS-L)(d-Ala)]⁺ monomers to form 1D hydrogen-bonded zigzag chains. The homochiral natures of complexes 1 and 2 have been confirmed by CD spectroscopy.     

Protonation Equilibria of Some Selected α-Amino Acids in DMSO–Water Mixture and Their Cu(II)-Complexes

The protonation constants of some α-amino acids (glycine (Gly), l-alanine (Ala), l-valine (Val), l-serine (Ser), l-leucine (Leu) and l-isoleucine (Ile)) were studied in water and DMSO–water solution mixtures containing 30, 50 and 70 vol-% DMSO; in addition the complex formation equilibria of their copper(II) complexes were studied by potentiometric technique using a combined pH electrode system calibrated in concentration units of the hydrogen ion at 25 ± 0.1 °C under a nitrogen atmosphere, and at an ionic strength of 0.10 mol·dm^?3 NaNO₃. The protonation constants and the overall stability constants of copper(II) complexes were influenced by changes in solvent composition, and their variations are discussed in terms of solvent and structural properties.     

l-Lactate Production from Seaweed Hydrolysate of Laminaria japonica Using Metabolically Engineered Escherichia coli

Renewable and carbon neutral, marine algal biomass could be an attractive alternative substrate for the production of biofuel and various biorefinery products. Thus, the feasibility of brown seaweed (Laminaria japonica) hydrolysate as a carbon source was investigated here for l-lactate production. This work reports the homofermentative route for l-lactate production by introducing Streptococcus bovis/equinus l-lactate dehydrogenase in an engineered Escherichia coli strain where synthesis of the competing by-product was blocked. The engineered strain utilized both glucose and mannitol present in the hydrolysate under microaerobic condition and produced 37.7 g/L of high optical purity l-lactate at 80 % of the maximum theoretical value. The result shown in this study implies that algal biomass would be as competitive with lignocellulosic biomass in terms of lactic acid production and that brown seaweed can be used as a feedstock for the industrial production of other chemicals.     

LC–MS–MS Simultaneous Determination of l-Dopa and Its Prodrug l-Dopa n-Pentyl Ester Hydrochloride in Rat Plasma

A sensitive, simple and rapid LC–MS–MS method has been developed and validated for the simultaneous determination of l-dopa and l-dopa n-pentyl ester hydrochloride in rat plasma in the present study. The analytes were separated on a C₁₈ column (5 μm, 2.1 × 150 mm) with a security guard C₁₈ column (5 μm, 4 × 20 mm) and a triple-quadrupole mass spectrometer was applied for detection. The method was linear over the concentration ranges of 25–5,000 ng mL^?1 for l-dopa and 12.5–2,500 ng mL^?1 for l-dopa n-pentyl ester hydrochloride. Finally, the method was successfully applied to support the pharmacokinetic study.     

Efficient Microbial Conversion of l-Tyrosine to l-DOPA by Brevundimonas sp. SGJ

l-DOPA (3,4-dihydroxyphenyl-l-alanine), the most widely used drug for the treatment of Parkinson??s disease, was produced in buffer using biomass of Brevundimonas sp. SGJ. The effects of enhancers, such as carrageenan, diatomaceous earth, and activated charcoal, on the l-DOPA production were evaluated to obtain the maximum yield. The optimal process conditions found were pH?8, 2?g?l^?1 cell mass, 2?g?l^?1 l-tyrosine, 0.04?g?l^?1 CuSO₄, 0.02?g?l^?1 l-ascorbic acid, 0.5?g?l^?1 carrageenan, and 40?°C temperature. In addition, repeated use of cells resulted in the highest yield of 3.81?g?l^?1 (95.2%) of l-DOPA with utilization of 4?g?l^?1 l-tyrosine, and the highest tyrosinase activity (9,201?U?mg^?1) was observed at 18?h of incubation. Furthermore, the produced l-DOPA was confirmed by high-performance thin-layer chromatography, high-performance liquid chromatography, and gas chromatography?Cmass spectroscopy. Kinetic studies showed significant values of Y _p/s, Q _s, and q _s after optimization of the process. Thus, Brevundimonas sp. SGJ could be an eventual new source for large-scale production of l-DOPA.     

Amperometric Detection of 3-(3,4-Dihydroxyphenyl)-l-alanine (l-Dopa) in Raw and Cooked Mucuna Bean Seeds Employing Micro-HPLC

Amperometric detection of 3-(3,4-dihydroxyphenyl)-l-alanine (l-dopa) on a glassy carbon electrode at oxidation potential of +0.70 V in Mucuna pruriens after micro-high performance liquid chromatography separation is reported. Optimised eluent consisted of 0.87 mM 1-octane sulphonic acid sodium salt, 18.2 mM citric acid, and 82.8 mM sodium acetate with pH adjusted to 2.18 using 85% orthophosphoric acid. Detection of low concentrations of l-dopa up to 5.12 ng mL^?1 was achieved. The method was employed to determine l-dopa in raw and cooked beans after water extraction through a 0.45 μm membrane with no further sample treatment.     

Xylitol Production by Genetically Engineered Trichoderma reesei Strains Using Barley Straw as Feedstock

Xylitol, a naturally occurring five-carbon sugar alcohol derived from d-xylose, is currently in high demand by industries. Trichoderma reesei, a prolific industrial cellulase and hemicellulase producing fungus, is able to selectively use d-xylose from hemicelluloses for xylitol production. The xylitol production by T. reesei can be enhanced by genetic engineering of blocking further xylitol metabolism in the d-xylose pathway. We have used two different T. reesei strains which are impaired in the further metabolism of xylitol including a single mutant in which the xylitol dehydrogenase gene was deleted (?xdh1) and a double mutant where additionally l-arabinitol-4-dehydrogenase, an enzyme which can partially compensate for xylitol dehydrogenase function, was deleted (?lad1?xdh1). Barely straw was first pretreated using NaOH and Organosolv pretreatment methods. The highest xylitol production of 6.1 and 13.22 g/L was obtained using medium supplemented with 2 % Organosolv-pretreated barley straw and 2 % d-xylose by the ?xdh1 and ?lad1?xdh1 strains, respectively.     

Comparison of ordered mesoporous materials sorption properties towards amino acids

The adsorption of amino acids such as l-phenylalanine and l-histidine was carried out on a series of mesoporous carbons obtained with the use ordered silicas KIT-6, SBA-16, SBA-15 as templates and furfuryl alcohol as carbon precursor. Small angle XRD analysis confirmed the ordered mesoporous structures of all materials obtained. They were also characterised by well-developed surface areas and high pore volumes. Adsorption behaviour of amino acids on ordered mesoporous carbons was investigated in potassium phosphate buffer solutions with adjustable l-phenylalanine and l-histidine concentration, ion strength, and pH. The highest sorption capacity towards the amino acids were observed at pH close to the isoelectric point of l-phenylalanine (pI = 5.48) and l-histidine (pI = 7.59). Electrostatic, hydrophobic and steric interactions had very strong effect on the adsorption of amino acids on mesoporous carbons. The amount of l-phenylalanine and l-histidine adsorbed decreased in the following sequence: C_KIT-6 > C_SBA-16 > C_SBA-15 that was strongly related to their structure, surface areas and average pore diameters.     

Extracellular l-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro

An extracellular l-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg^?1. The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified l-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K _m, V _max, and k _cat values of the enzyme were 0.257 mM, 1.537 U μg^?1, and 993.93 s^?1, respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α?+?β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified l-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the l-asparaginase has significant antineoplastic properties.     

RP-LC Resolution of (R,S)-Atenolol via Diastereomerization with Marfey’s Reagent and Its Structural Variants Under Conventional and Microwave Heating

(R,S)-Atenolol was derivatized with Marfey’s reagent, (MR; 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide or FDNP-l-Ala-NH₂) and its four structural variants (FDNP-l-Phe-NH₂, FDNP-l-Val-NH₂, FDNP-l-Leu-NH₂ and FDNP-l-Pro-NH₂). MR reacts quantitatively with 1° and 2° amino groups and atenolol has a secondary amino group. The derivatization reactions were carried out under conventional and microwave heating and compared. The resulting diastereomers were separated on RP-TLC and on a C18 column with detection at 340 nm. (R)-Isomer eluted before (S). The conditions of derivatization and chromatographic separation were optimized. The method was validated for linearity, repeatability, limits of detection and limit of quantification.     

An Improved and Validated LC Method for Resolution of Panthenol

An improved LC method was developed and validated for determination of enantiomeric purity of panthenol in bulk drugs. The method is based on derivatization of panthenol with 3,5-dinitrobenzoyl chloride. Baseline separation with resolution >2.7 was achieved within 20 min on Kromasil CHI-DMB (250 × 4.6 mm) column using n-hexane:ethanol (95:5 v/v) as mobile phase at a flow rate of 1.5 mL min^?1. The analytes were detected by their UV absorbance at 265 nm. The effects of ethanol, 2-propanol and temperature on enantioselectivity and resolution of enantiomers were evaluated. The method was extensively validated and proved to be robust. The recoveries were between 98.3 and 101.4% with <1.6% relative standard deviation. The regression equations for the derivatives of d-panthenol and l-panthenol were y ₁ = 18.01x ₁ ? 32.56 (r ₁ ²  = 0.9984) and y ₂ = 17.855x ₂ ? 28.16 (r ₂ ²  = 0.9990), respectively. The LOD and LOQ for the derivative of d-panthenol were 10.6 and 37.4 μg mL^?1 and for the derivative of l-panthenol were 12.1 and 40 μg mL^?1, respectively. The improved method was found to be simple, rapid, and sensitive for the determination of enantiomeric purity of panthenol in bulk drugs.     

Interactions of some protonated α-amino acid methyl esters with hexaethyl p-tert-butylcalix[6]arene hexaacetate in nitrobenzene saturated with water

The exchange extraction constants corresponding to the general equilibrium C⁺(aq) + 1·Cs⁺(nb) ? 1·C⁺ (nb) + Cs⁺(aq) occurring in the two-phase water–nitrobenzene system (C⁺ = protonated α-amino acid methyl ester, 1 = hexaethyl p-tert-butylcalix[6]arene hexaacetate; aq = aqueous phase, nb = nitrobenzene phase) were evaluated on the basis of extraction experiments and γ-activity measurements. Further, the stability constants of the 1·C⁺ cationic complex species in nitrobenzene saturated with water were calculated; they were found to increase in the following cation order: protonated l-tryptophan methyl ester < protonated l-phenylalanine methyl ester < protonated l-leucine methyl ester < protonated l-methionine methyl ester < protonated l-valine methyl ester.     

Enhanced l-Lactic Acid Production from Biomass-Derived Xylose by a Mutant Bacillus coagulans

Xylose effective utilization is crucial for production of bulk chemicals from low-cost lignocellulosic substrates. In this study, an efficient l-lactate production process from xylose by a mutant Bacillus coagulans NL-CC-17 was demonstrated. The nutritional requirements for l-lactate production by B. coagulans NL-CC-17 were optimized statistically in shake flask fermentations. Corn steep liquor powder and yeast exact were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors, and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform batch fermentation in a 3-l bioreactor. A maximum of 90.29 g l^?1? l-lactic acid was obtained from 100 g l^?1 xylose in 120 h. When using corn stove prehydrolysates as substrates, 23.49 g l^?1? l-lactic acid was obtained in 36 h and the yield was 83.09 %.     

Sensitivity of the Multiple Functional Moieties of Amino Acids for the Self-Assembly of Au Nanoparticles on Different Physicochemical Properties

This paper investigates the extent of the self-assembly process of Au nanoparticles, depending on the nature of structural and functional moieties of various amino acids (l-cystine, glutathione, l-cysteine and N-acetyl cysteine) and their influence on the plasmon sensitivity and electrokinetic parameters in correlation with the catalysis of p-nitrophenol reduction. DLS particle size analysis revealed that the hydrodynamic size 10–20 nm of Au nanospheres was increased to 135–550 nm, 100–460 nm and 130–240 nm after the addition of l-cystine, l-cysteine and glutathione, respectively, in contrast to no significant change of particle size (15–60 nm) after N-acetyl cysteine addition. This difference in the extent of aggregation as a function of structures of amino acids is further evidenced by lengthy tubular arrays formation by glutathione as compared to branched chain like morphology obtained by l-cystine through TEM. FTIR studies further confirmed the binding of amino acids to Au nanospheres via –SH followed by linking of adjacent nanoparticles through H-bonding. Due to the conformational diversity of amino acids, the surface adsorbed –SH, –COO^? and –NH₃ ⁺ species over assembled Au nanoparticles led to the alteration of zeta potential and conductance, thus affected the catalysis for the reduction of p-nitrophenol as compared to unmodified Au nanoparticles.     

Thermal properties and interactions of l-proline in aqueous solutions of NaCl or KCl at different temperatures

The enthalpies of solution of l-proline in aqueous electrolyte solutions within the electrolyte molality range up to 4.9 mol kg^?1 of NaCl and up to 4.0 mol kg^?1 of KCl at 288, 298 and 313 K have been measured by the calorimetric method. Enthalpies of transfer of l-proline from water to aqueous electrolyte solutions up to saturation have been derived at 273–348 K. The enthalpic and heat capacity parameters of pair and triplet interaction of l-proline with electrolyte in water have been evaluated. Enthalpic parameters of pair interaction at 298 K have been compared to similar parameters for glycine and l-alanine. The temperature changes of reduced enthalpy, and also the change of entropy and reduced Gibbs energy of transfer of l-proline from water to aqueous electrolyte solution at temperature rise from 273 to 323 K have been determined. It has been shown that the entropy–enthalpy compensation takes place for transfer processes.     

Enhancing l-Isoleucine Production by thrABC Overexpression Combined with alaT Deletion in Corynebacterium glutamicum

l-isoleucine is synthesized from 2-ketobutyrate and pyruvate in Corynebacterium glutamicum, and the supplies of these two precursors are important for l-isoleucine synthesis. C. glutamicum YILWΔalaT with alaT gene deletion (encoding alanine aminotransferase, a principal enzyme for l-alanine synthesis) was constructed to increase intracellular pyruvate availability, and the thrABC genes from Escherichia coli (encoding bifunctional aspartate kinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase) were overexpressed in C. glutamicum YILW and YILWΔalaT to increase the supply of intracellular 2-ketobutyrate. In the fed-batch fermentation, YILWpXMJ19thrABC, YILWΔalaT, and YILWΔalaTpXMJ19thrABC exhibited 5.3, 17.6, and 8.4 % higher l-isoleucine production than the original strain, respectively. Both YILWpXMJ19thrABC and YILWΔalaT excreted lower concentrations of l-lysine, l-alanine, and l-valine. YILWΔalaTpXMJ19thrABC exhibited a cumulative reduction of these by-products excretion, which indicated that thrABC overexpression combined with alaT deletion resulted in the metabolic flux redistribution from 2-ketobutyrate and pyruvate to l-isoleucine synthesis, and decreased the fluxes to by-products synthesis accordingly.