Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Identifying Key Amino Acid Residues That Affect α-Conotoxin AuIB Inhibition of α3β4 Nicotinic Acetylcholine Receptors

α-Conotoxin AuIB is a selective α3β4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3β4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3β4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3β4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3β4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the β4-Trp-59 and β4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the β4 subunit and gave further insight into how AuIB Phe-9 interacts with the β4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively "avoid" nAChR receptors while targeting receptors involved with nociception.

ICH guideline practice: application of novel RP-HPLC-DAD method for determination of olopatadine hydrochloride in pharmaceutical products

BackgroundA novel reverse-phase high-performance liquid chromatography (RP-HPLC)-DAD method was developed, validated and applied to quantify olopatadine hydrochloride in pharmaceutical products.MethodsThe RP-HPLC analyses were carried out using a mixture of 0.1% formic acid and methanol (35:65) as a mobile phase on ZORBAX Eclipse Plus C18 (250 mm × 4.6 mm, 5 μm), with a flow rate of 1.0 mL/min and UV detection at 300 nm. The developed method was validated as per the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceutical for Human Use guidelines for linearity, range, accuracy, precision, robustness, limit of detection, limit of quantitation and specificity.ResultsThe validated method was applied to quantify the drug content in raw material, tablets and eye drops which was found to be 99.36% to 101.02%, 98.99% to 100.56% and 99.09% to 100.91%, respectively.ConclusionThe method was found to be efficient, precise, accurate, specific and economic and useful for the routine analysis of olopatadine hydrochloride in pharmaceutical industries.

Elution strategies for reversed-phase high-performance liquid chromatography analysis of sucrose alkanoate regioisomers with charged aerosol detection

A broad range of elution strategies for RP-HPLC analysis of sucrose alkanoate regioisomers with CAD was systematically evaluated. The HPLC analyses were investigated using design-of-experiments methodology and analysed by analysis of variance (ANOVA) and regression modelling. Isocratic elutions, isocratic elutions with increased flow, and gradient elutions with step-down profiles and step-up profiles were performed and the chromatographic parameters of the different elution strategies were described by suitable variables. Based on peak resolutions general resolution deviation for multiple peaks (RDm) was developed for sample-independent evaluation of separation of any number of peaks in chromatographic analysis. Isocratic elutions of sucrose alkanoates showed similar relationships between eluent acetonitrile concentration and retention time for all regioisomers of sucrose caprate and sucrose laurate, as confirmed by evaluation of the curvatures using approximate second derivatives and Kendall rank correlation coefficients. Regression modelling and statistical analysis showed that acetonitrile concentration and flow rate were highly significant for both average adjusted retention time and RDm for sucrose laurate. For both responses the effect of changes in acetonitrile concentration was larger than the effect of changes in flow rate, over the ranges studied. Regression modelling of the step-down gradient profiles for the sucrose alkanoates showed that the eluent acetonitrile concentrations were the overall most significant variables for retention time and separation. The models for average adjusted retention time of sucrose caprate and sucrose laurate showed only a few differences in the significance levels of terms, while the models for RDm showed larger differences between the sucrose alkanoates, in both the number of terms and their significance. Efficiency evaluation of elution strategies, in terms of RDm and analysis time, showed that the best results were offered by step-down gradient elution for sucrose caprate and isocratic elution with increased flow for sucrose laurate. Step-down gradient elution of sucrose caprate offered improvements in separation at similar analysis time compared to isocratic elution, with the most efficient elutions achieved with elution profile acetonitrile concentrations at 32.5% and 25%, resulting in reduction of RDm by 13–38% and reduction of analysis time by 3–9%. For sucrose laurate, isocratic elution with increased flow showed improvements in separation and reductions in analysis time compared to isocratic elution, such as elution at 37% with flow 2.0mL/min resulting in reduction of analysis time by 34% and equal RDm, while elution at 35% with flow 2.0mL/min reduced RDm by 29% and analysis time by 6%, compared to isocratic elution at 38% acetonitrile with standard flow (1.0mL/min).

Specific Activation of the G Protein-coupled Receptor BNGR-A21 by the Neuropeptide Corazonin from the Silkworm, Bombyx mori, Dually Couples to the Gq and Gs Signaling Cascades

Corazonin, an undecapeptide neurohormone sharing a highly conserved amino acid sequence across Insecta, plays different physiological roles in the regulation of heart contraction rates, silk spinning rates, the induction of dark color and morphometric phase changes, and ecdysis. Corazonin receptors have been identified in Drosophila melanogaster, Manduca sexta, and Musca domestica. However, detailed information on the signaling and major physiological functions of corazonin and its receptor is largely unknown. In the current study, using both the mammalian cell line HEK293 and insect cell lines BmN and Sf21, we paired the Bombyx corazonin neuropeptide as a specific endogenous ligand for the Bombyx neuropeptide G protein-coupled receptor A21 (BNGR-A21), and we therefore designated this receptor as BmCrzR. Further characterization indicated that synthetic BmCrz demonstrated a high affinity for and activated BmCrzR, resulting in intracellular cAMP accumulation, Ca(2+) mobilization, and ERK1/2 phosphorylation via the Gq- and Gs-coupled signaling pathways. The direct interaction of BmCrzR with BmCrz was confirmed by a rhodamine-labeled BmCrz peptide. Moreover, experiments with double-stranded RNA and synthetic peptide injection suggested a possible role of BmCrz/BmCrzR in the regulation of larval growth and spinning rate. Our present results provide the first in-depth information on BmCrzR-mediated signaling for further elucidation of the BmCrz/BmCrzR system in the regulation of fundamental physiological processes.

Preparation of Highly Concentrated Influenza Vaccine for Use in Novel Delivery Approaches

Vaccine antigens are usually available only as dilute solutions, which are difficult to formulate into various novel delivery systems, which often require highly concentrated antigens. To address this problem, we have utilized tangential flow filtration (TFF), a simple and scalable process to prepare highly concentrated vaccine antigens. Here, we describe the optimization of TFF to concentrate hemagglutinin (HA) of egg-derived influenza antigens, from 2008 to 2009 seasonal vaccine, to concentrations up to 28 mg/mL. Concentrated antigen was evaluated by single radial immunodiffusion and reversed-phase high-performance liquid chromatographic analysis for the estimation of the HA content and a range of assays including size exclusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and bicinchoninic acid assay for protein characterization. In addition, the concentrated antigens retained their immunogenicity, confirmed by the induction of immune responses comparable to that of unprocessed antigen in a mouse model. The liquid concentrates were stable for up to 4 weeks, which could allow subsequent formulation into novel delivery technologies. Hence, we have used influenza HA to demonstrate that the fast, robust, and scalable approach of TFF can be used to concentrate antigens for use in novel delivery approaches. Moreover, the concentration process could be applicable for a variety of antigens and a wide range of novel vaccine delivery applications.

Assessment of adrenocortical activity by non-invasive measurement of faecal cortisol metabolites in dromedary camels (Camelus dromedarius)

The aim of this study was to determine whether glucocorticoid production could be monitored non-invasively in dromedary camels by measuring faecal cortisol metabolites (FCMs). Five Sudanese dromedaries, two males and three females, were injected with a synthetic adrenocorticotropic hormone (ACTH) analogue. Blood samples were collected pre- and post-ACTH injection. Faeces were sampled after spontaneous defecation for five consecutive days (2 days before and 3 days after ACTH injection). Baseline plasma cortisol values ranged from 0.6 to 10.8 ng/ml in males and from 1.1 to 16.6 ng/ml in females, while peak values after ACTH injection were 10.9-41.9 in males and 10-42.2 ng/ml in females. Peak blood cortisol values were reached between 1.5 and 2.0 h after ACTH injection. The concentration of FCMs increased after ACTH injection in the faeces of both sexes, although steroid levels peaked earlier in males [24 h; (286.7-2,559.7 ng/g faeces)] than in females [36-48 h; (1,182.6-5,169.1 ng/g faeces)], reflecting increases of 3.1-8.3- and 4.3-8-fold above baseline levels. To detect chromatographic patterns of immunoreactive FCMs, faecal samples with high FCM concentrations from both sexes were pooled and subjected to reverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC analysis revealed sex differences in the polarity of FCMs, with females showing more polar FCMs than males. We concluded that stimulation of adrenocortical activity by ACTH injection resulted in a measurable increase in blood cortisol that was reliably paralleled by increases in FCM levels. Thus, measurement of FCMs is a powerful tool for monitoring the adrenocortical responses of dromedaries to stressors in field conditions.

Purification and hypotensive activity of rapeseed protein-derived renin and angiotensin converting enzyme inhibitory peptides

Rapeseed protein isolate (RPI) was hydrolyzed with Alcalase followed by reverse-phase high performance liquid chromatography (RP-HPLC) purification of bioactive peptides. The rapeseed protein hydrolysate (RPH) obtained after 4h digestion with Alcalase had a degree of hydrolysis (DH) of ∼11%. Gel permeation chromatography separation showed high contents of low molecular weight peptides in the RPH when compared to the RPI. After preparative and analytical RP-HPLC separations, three peptides (LY, TF and RALP) were purified and amino acid sequence determined by tandem mass spectrometry. LY (IC50, 0.11mM) was the most potent (p<0.05) against ACE activity when compared to TF (IC50, 0.81mM) and RALP (IC50, 0.65mM). However, RALP (IC50, 0.97mM) was the most potent (p<0.05) against renin activity when compared to LY (IC50, 1.87mM) and TF (IC50, 3.1mM). Single oral administration (30mg/kg body weight) to spontaneously hypertensive rats showed LY and RALP to be the more effective hypotensive agents with maximum blood pressure reduction of −26 and 16mmHg, respectively when compared to TF (−12mmHg). The results suggest that the higher number of hydrophobic amino acid residues LY and RALP contributed to their higher in vitro and in vivo activities when compared to TF.

Selective HPLC method development for soy phosphatidylcholine fatty acids and its mass spectrometry

A novel, efficient and simple approach for soy phosphatidylcholine analysis according to its fatty acid composition was studied with reverse-phase high-performance liquid chromatography. The reverse-phase high-performance liquid chromatography analysis was performed isocratically using UV detector and simple mobile phase solvents consisting of isopropyl alcohol, methanol, and deionized water in the proportion of 70:8:22 v/v. The uniqueness of the proposed method was the separation of individual fatty acids of soy phosphatidylcholine. The high-performance liquid chromatography method for soy phosphatidylcholine was validated for linearity with correlation coefficient of above 0.99 for all the peaks separated according to their fatty acid composition. The intra-day and the inter-day precision studies provided the relative standard deviation of less than 2%. The limit of detection and limit of quantitation values were also calculated for all the resolved peaks of soy phosphatidylcholine. Also system performance parameters such as number of theoretical plates, capacity factor, tailing factor, separation factor, and peak resolution were studied systematically and found well within the acceptable range. The proposed high-performance liquid chromatography method was successfully applied to soy phosphatidylcholine extracted and purified from deoiled soy lecithin without any interference of impurities or solvent peaks. Individually, the collected peaks of sample soy phosphatidylcholine were subjected for mass spectroscopy. The mass spectra showed all the peaks having different saturated or unsaturated fatty acid chains attached to glyerophosphocholine moiety of soy phosphatidylcholine. The method developed is economic and well suited for estimation of soy phosphatidylcholine with its fatty acid composition.

An alkaline protease from Kocuria kristinae F7: properties and characterization of its hydrolysates from soy protein

An alkaline serine protease (SF1) from Kocuria kristinae F7 was purified. The molecular mass of the SF1 protease was estimated to be 57 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The N-terminal amino acid sequence of the first 14 amino acids of the SF1 protease showed low homology with bacterial proteases, suggesting that the enzyme had not been described previously. The SF1 protease exhibited maximal activity at pH 9.0 and 60 °C. The activity of the SF1 protease was enhanced by the presence of Ca2+ and Mg2+ ions. It showed high stability in the presence of NaCl and ethanol. Reverse-phase high-performance liquid chromatography (RP-HPLC) analyses indicated that soybean protein isolates treated with the SF1 protease generated four principal new hydrophilic peptides. Mass spectrometry analyses indicate that the distribution of molecular weight of these peptides was from 0.705 to 1.305 kDa. Hydrolysis of soybean protein isolates with the SF1 protease increased the level of total free amino acids, essential amino acids and flavor amino acids. The SF1 protease may decrease the bitterness of soy protein hydrolysates. The results showed that the SF1 protease of Kocuria kristinae F7 appears to be good candidate enzyme for potential application in acceleration of fermented soybean food ripening.

Phytochemistry and bioactivity of Pedicularis sibthorpii growing in Iran

The methanol extract of the aerial parts of the medicinal plant Pedicularis sibthorpii Boiss., Scrophulariaceae, growing in the Azerbaijan province of Iran, was found to be active in the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and the antibacterial agar well diffusion assays, but no general toxicity was observed in the brine shrimp lethality assay. A combination of solid-phase extraction (SPE) and preparative reversed-phase high-performance liquid chromatography (prep-RP-HPLC) analyses of the methanolic extract afforded three phenylethanoids (verbascoside, martynoside and isomartynoside), an iridoid (aucubin), a flavonoid (luteolin 7-O-β-D-glucopyranoside) and mannitol, and the structures of these compounds were elucidated unambiguously by spectroscopic means. The distribution of the isolated compounds within the genus Pedicularis has also been discussed.

Examination of the genome-wide transcriptional response of Escherichia coli O157:H7 to cinnamaldehyde exposure.

Cinnamaldehyde is a natural antimicrobial that has been found to be effective against many food-borne pathogens, including Escherichia coli O157:H7. Although its antimicrobial effects have been well investigated, limited information is available on its effects at the molecular level. Sublethal treatment at 200 mg/liter cinnamaldehyde inhibited growth of E. coli O157:H7 at 37°C and for ≤2 h caused cell elongation, but from 2 to 4 h growth resumed and cells reverted to normal length. To understand this transient behavior, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h of exposure to cinnamaldehyde in conjunction with reverse-phase high-performance liquid chromatography (RP-HPLC) analysis for cinnamaldehyde and other cinnamic compounds. Drastically different gene expression profiles were obtained at 2 and 4 h. RP-HPLC analysis showed that cinnamaldehyde was structurally stable for at least 2 h. At 2 h of exposure, cinnamaldehyde induced expression of many oxidative stress-related genes and repressed expression of DNA, protein, O-antigen, and fimbrial synthetic genes. At 4 h, many cinnamaldehyde-induced repressive effects on E. coli O157:H7 gene expression were reversed, and cells became more motile and grew at a slightly higher rate. Data indicated that by 4 h, E. coli O157:H7 was able to convert cinnamaldehyde into the less toxic cinnamic alcohol using dehydrogenase/reductase enzymes (YqhD and DkgA). This is the first study to characterize the ability of E. coli O157:H7 to convert cinnamaldehyde into cinnamic alcohol which, in turn, showed that the antimicrobial activity of cinnamaldehyde is mainly attributable to its carbonyl aldehyde group.

RP-HPLC analysis for the simultaneous estimation of rabeprazole sodium and aceclofenac in a combined dosage form

A rapid, simple and highly sensitive reversed phase high performance liquid chromatographic (RP-HPLC) method has been developed for the quantitative determination of Rabeprazole sodium and Aceclofenac in a combined dosage form. Rabeprazole sodium and Aceclofenac were chromatographed using C-18 column as stationary phase and methanol: acetonitrile: water (60 : 10 : 30 v/v/v) as the mobile phase at a flow rate of 1.0 ml/min at ambient temperature and detected at 280 nm. The retention time (RT) of Rabeprazole sodium and Aceclofenac were found to be 5.611 min and 2.102 minute, respectively. The linearities of Rabeprazole sodium and Aceclofenac were in the range of 1-10 µg/ml and 3-15 µg/ml, respectively. The limit of detection was found to be 0.091 µg/ml for Rabeprazole sodium and 0.043 µg/ml for Aceclofenac. The proposed method was applied for the determination of Rabeprazole sodium and Aceclofenac in a combined dosage form and result was found satisfactory.DOI: http://dx.doi.org/10.3329/icpj.v1i12.12450 International Current Pharmaceutical Journal 2012, 1(12): 410-413

Biocontrol of green and blue molds in postharvest satsuma mandarin using Bacillus amyloliquefaciens JBC36

In order to control postharvest rot of satsuma mandarin (mandarin) fruits, a new strain of Bacillus amyloliquefaciens JBC36 (JBC36) was isolated from rhizosphere of a mandarin orchard and tested for its suppression of decay due to green and blue molds caused by Penicillium digitatum and Penicillium italicum, respectively, and its mode of action was investigated. In addition, carnauba wax-based and paraffin oil-based coating formulations were developed to increase the control efficacy of the antagonist. The strain JBC36 at 108 CFU mL−1 inhibited incidence of green and blue molds on wounded mandarin fruits with control efficacies of 88 and 80.2%, respectively. Mycelial growth and spore germination of P. digitatum and P. italicum were strongly inhibited in the presence of JBC36 or antagonistic metabolites. In order to determine antifungal activity, three kinds of antibiotics were isolated by reverse-phase high performance liquid chromatography (RP-HPLC) and identified as lipopeptide families, iturin A, fengycin and surfactin by RP-HPLC and thin-layer chromatography (TLC) analysis. Fengycin was further identified as C16 fengycin A by liquid chromatography/electrospray ionisation-mass spectrometry (LC/ESI-MS) and mass spectrometry/mass spectrometry (MS/MS) analysis. Volatile organic compounds from the antagonist also reduced the mycelial growth of P. digitatum and P. italicum. Carnauba wax-based and paraffin oil-based coating formulations containing 108 CFU mL−1 of JBC36 efficiently decreased the incidence of green mold with control efficacy of 91 and 80.9%, respectively. Overall, the antagonistic rhizobacterium JBC36 is a promising biocontrol agent for use in preventing postharvest spoilage of mandarin fruits by green and blue molds.

Reversed Phase HPLC Analysis of Stability and Microstructural Effects on Degradation Kinetics of β-Carotene Encapsulated in Freeze-Dried Maltodextrin–Emulsion Systems

Degradation of dispersed lipophilic compounds in hydrophilic solids depends upon matrix stability and lipid physicochemical properties. This study investigated effects of solid microstructure and size of lipid droplets on the stability of dispersed β-carotene in freeze-dried systems. Emulsions of β-carotene in sunflower oil were dispersed in maltodextrin systems (M040/DE6, M100/DE11, and M250/DE25.5) (8% w/w oil) and prefrozen at various freezing conditions prior to freeze-drying to control nucleation and subsequent pore size and structural collapse of freeze-dried solids. The particle size, physical state, and β-carotene contents of freeze-dried emulsions were measured during storage at various water activity (a(w)) using a laser particle size analyzer, differential scanning calorimeter, and high performance liquid chromatography (HPLC), respectively. The results showed that M040 stabilized emulsions in low temperature freezing exhibited lipid crystallization. Collapse of solids in storage at a(w) which plasticized systems to the rubbery state led to flow and increased the size of oil droplets. Degradation of β-carotene analyzed using a reversed-phase C(30) column followed first-order kinetics. Porosity of solids had a major effect on β-carotene stability; however, the highest stability was found in fully plasticized and collapsed solids.

Changes in prolamin and high resistant starch composition during the production process of Boza, a traditional cereal-based beverage

The aim of this work was to analyze changes occurring in cereal protein and starch composition throughout the production process of Boza for its potential exploitation as functional beverage. A sharp decrease in the extractability of albumins, globulins, glutenins and prolamins was found in the processed samples after the cooking step. This phenomenon was confirmed analyzing the extracted cereal proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which also revealed an apparent decrease in the number of wheat albumin/globulin, glutenin and prolamin bands in the gruel samples in comparison with that shown by the related flours. The application of an improved extraction procedure and reverse-phase high-performance liquid chromatography analysis confirmed that the prolamin content in Boza decreased markedly in comparison with that of the flour samples. Under the experimental conditions applied, the resistant starch content of Boza resulted significantly higher than that of flours; thus, this beverage can be regarded as a “high fiber” food in accordance with the recent European regulations. This is the first report that throws light on changes occurring in protein and starch components throughout the production process of Boza and provides direct evidence on the low prolamin and high resistant starch contents of this traditional cereal-based beverage.

Identification and Characterization of Buried Unpaired Cysteines in a Recombinant Monoclonal IgG1 Antibody

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.

Biodegradation of the Anionic Surfactant Linear Alkylbenzene Sulfonate (LAS) by Autochthonous Pseudomonas sp.

Anionic surfactants, the earliest and the most common surfactants in detergent and cosmetic product formulations contribute significantly to the pollution profile of the ecosystem. Linear alkylbenzene sulfonates (LAS), a major chemical constituent of detergents, forms an imperative group of anionic surfactants. Bioremediation of LAS by conventional processes such as activated sludge is ineffective due to the low kinetics of degradation by unsuitable organisms and foam production. Hence this study was focused on isolating and characterizing indigenous LAS-degrading bacteria from soil. Twenty different LAS-degrading bacteria were isolated from detergent-contaminated soil by enrichment culture technique and degradation efficiency was assessed by Methylene Blue Active Substances (MBAS) assay and by reverse-phase high-performance liquid chromatography (HPLC) analysis. The most efficient LAS-degrading isolates, L9 (81.33 ± 0.7) and L12 (81.81 ± 0.8), were selected and identified as Pseudomonas nitroreducens (MTCC 10463) and Pseudomonas aeruginosa (MTCC 10462). The 16S rDNA sequences of the isolates were deposited in NCBI GenBank under the accession numbers HQ 271083 (L9) and HQ 271084 (L12). The isolates were capable of degrading 0.05 g/l LAS at 25 °C and pH 7.0–7.5. Presence of a solid support caused biofilm formation which in turn enhanced LAS degradation. The isolates tend to display diauxic growth with alternate carbon source such as dextrose. These isolates also have the capability to degrade other xenobiotics like hydrocarbons and pesticides. Since xenobiotic pollutants in nature occur as a mixture of compounds rather than single pollutants, the potential of these two indigenous LAS-degrading isolates to degrade multiple xenobiotics gains relevance.

Comparison of UV spectrophotometry and high performance liquid chromatography methods for the determination of repaglinide in tablets

Background: Repaglinide is a miglitinide class of antidiabetic drug used for the treatment of type 2 diabetes mellitus. A fast and reliable method for the determination of repaglinide was highly desirable to support formulation screening and quality control. Objective: UV spectrophotometric and reversed-phase high performance liquid chromatography (RP-HPLC) methods were developed for determination of repaglinide in the tablet dosage form. Materials and Methods: The UV spectrum recorded between 200 400 nm using methanol as solvent and the wavelength 241 nm was selected for the determination of repaglinide. RP-HPLC analysis was carried out using AgilentTC-C18 (2) column and mobile phase composed of methanol and water (80:20 v/v, pH adjusted to 3.5 with orthophosphoric acid) at a flow rate of 1.0 ml/min. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the International Conference on Harmonization (ICH) guidelines. Results: The developed methods illustrated excellent linearity (r2 > 0.999) in the concentration range of 5–30 μag/ml and 5–50 μag/ml for UV spectrophotometric and HPLC methods, respectively. Precision (%R.S.D < 1.50) and mean recoveries were found in the range of 99.63-100.45% for UV spectrophotometric method and 99.71100.25% for HPLC method which shows accuracy of the methods. Conclusion: The developed methods were found to be reliable, simple, fast, accurate and successfully used for the quality control of repaglinide as a bulk drug and in pharmaceutical formulations.

Development and validation of a useful HPLC–UV method for quantification of total and phosphorylated-ribavirin in blood and erythrocytes of HCV+ patients

Ribavirin-induced hemolytic anemia is the main cause of discontinuation of the combination therapy with alpha-interferon-2b and ribavirin for the treatment of hepatitis C virus (HCV) infection. The determination of intracellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, which could be caused by the accumulation of these molecules within the erythrocytes. In this work, we simplified and validated a previously developed assay method, to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a five-fold volume of ice-cold distilled underwent a process of acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated with a protein precipitation protocol in acetonitrile, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations from 625 to 320,000 ng/mL (r(2)=0.998). Accuracy, intra-day and inter-day precision for ribavirin and phosphorylated-ribavirin quality controls were all below 9.0%. We tested this method by monitoring blood ribavirin concentrations in 13 HCV+ patients, receiving alpha interferon-plus ribavirin combination therapy.

Thermal behavior of malonylglucoside isoflavones in soybean flour analyzed by RPHPLC/DAD and eletrospray ionization mass spectrometry

Soybean (Glycine max (Merrill) L.) contains high content of aglycone isoflavones, as well as glucoside and malonylconjugates. In this work, the content of isoflavones in defatted soy flour was determined by reversed-phase high-performance liquid chromatography (RPHPLC) after alcoholic extraction in methanol/water mixture in the ratio 80:20 (v/v). It was observed that the heating treatment transformed the malonylglucosides into glucoside isoflavones. After heat treatment at 121°C for 30min, nearly all malonylisoflavones were converted into glucoside, but acetylisoflavones were not detected via RPHPLC analysis. Electrospray ionization mass spectrometry confirmed the presence of malonylisoflavones in heat-treated defatted soy flour by direct infusion analysis.