A validated stability-indicating HPLC method for the determination of PEGylated puerarin in aqueous solutions

The aim of this study was to develop a validated specific stability-indicating HPLC method for the quantitative determination of PEGylated puerarin (PEG-PUE) in aqueous solutions. The method was validated by subjecting PEG-PUE to forced degradation under stress conditions of acid, alkali, water hydrolysis, and oxidation. Both PEG-PUE and puerarin (PUE) were simultaneously determined and separated on CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1.0 mL min⁻¹ and detection wavelength was set at 250 nm. Both calibration curves showed good linear regression (r ≥ 0.9998) within test ranges. The LOD and LOQ of PEG-PUE were determined to be 3 and 9 μg mL⁻¹ respectively. Degradation of PEG-PUE followed pseudo-first-order kinetics with t_1/2 of 59 min at pH 9.0 and 17.79 h at pH 7.4. However, at pH 5.0 and 2.0, there was no significant degradation of PEG-PUE over time. In conclusion, the method was observed to have the necessary specificity, precision, and accuracy, and to be suitable for quantity monitoring the degradation process of PEG-PUE during stability studies. The degradation studies may give insight into useful information for formulation development of PEG-PUE.     

Development of an effective novel validated stability-indicating HPLC method for the resolution of brivaracetam stereoisomeric impurities

Reverse-phase high-performance liquid chromatography method has been developed for the determination of brivaracetam stereoisomeric impurities such as (R,S)-brivaracetam, (R,R)-brivaracetam, and (S,S)-brivaracetam with good resolution using the chiral column, Chiral PAK IG-U (100 × 3.0 mm; 1.6 μm). The method is simple, stability-indicating, and compatible with LC–MS. The separation was achieved with the mobile phase consisted of 10 mM ammonium bicarbonate along with acetonitrile in an isocratic mode. The column temperature and wavelength were monitored at 40°C and 215 nm, respectively. The method showed adequate specificity, sensitivity, linearity, accuracy, precision, and robustness inline to ICH tripartite guidelines. The limit of detection and quantification limits were 0.3 and 0.8 μg ml⁻¹, respectively, for all stereoisomeric impurities and brivaracetam. The developed method was found to be linear over the concentration range of 0.8–5.6 μg ml⁻¹ for stereoisomeric impurities with a correlation coefficient >0.999. The method was precise (%RSD < 5.0), robust, and accurate (with 85%–115% recovery). The values of retention times of stereoisomeric impurities, (R,S)-brivaracetam, (R,R)-brivaracetam, and (S,S)-brivaracetam, were 4.9, 5.4, and 6.6 min, respectively, and resolution among the impurities were 2.0, 3.3, and 4.7, respectively. In addition, forced degradation studies were performed to prove that method was stability-indicating. The enrichment of isomeric impurity, (R,R)-brivaracetam, was observed under basic stress conditions of brivaracetam and proposed a plausible mechanism to enhance that isomeric impurity. As well, a good separation among brivaracetam and its stereoisomeric impurity peaks was observed in the presence of degradation products and process-related impurities.     

A validated enantiospecific method for determination and purity assay of clopridogrel

A new and accurate HPLC method using β‐cyclodextrin chemically bonded to spherical silica particles as chiral stationary phase (CSP) was developed and validated for determination of S‐clopidogrel and its impurities R‐enantiomer and S‐acid as a hydrolytic product. The effects of acetonitrile and methanol content in the mobile phase and temperature on the resolution and retention of enantiomers were investigated. A satisfactory resolution of S‐clopidogrel active form and its impurities was achieved on ChiraDex® column (5 μm, 4 × 250 mm) at a flow rate of 1.0 ml/min and 17°C using acetonitrile, methanol and 0.01 M potassium dihydrogen phosphate solution (15:5:80 v/v/v) as mobile phase. The detection wavelength was set at 220 nm. The method was validated in terms of accuracy, precision, linearity, and robustness. The limit of detection for R‐enantiomer and S‐acid were 0.75 and 0.09 μg/ml, respectively, injection volume being 20 μl. Finally, the molecular modeling of the inclusion complexes between the analytes and β‐cyclodextrin was performed to investigate the mechanism of the enantiorecognition and to study the quantitative structure–retention relationships. Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

Behavior of endothelial cells on Matrigel and development of a method for a rapid and reproducible in vitro angiogenesis assay

During the process of angiogenesis, the normally quiescent endothelial cells that line the vasculature are induced to proliferate, migrate and align to form new blood vessels by angiogenic stimuli. Assays for angiogenic factors mostly involve in vivo approaches. The two most commonly used in vivo assays—the chick chorioallantoic membrane (CAM) assay and the rabbit corneal assay are tedious to perform and are technically demanding. Several in vitro assays have also been developed, based on the ability of endothelial cells to form tubes in 3-D matrices. Here, we describe the modification of a microcarrier bead-based assay. This assay combines cells grown on Cytodex-3 microcarrier beads with Matrigel to provide an easy, rapid, and reliable method for evaluating and measuring angiogenic activity. We also describe the differential behavior of normal and transformed endothelial cells cultured in Matrigel.     

Total antioxidant performance: a validated fluorescence assay for the measurement of plasma oxidizability

The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750mug/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, alpha-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid (R=0.97, P<0.05) and cholesterol-adjusted alpha-tocopherol (R=0.93, P<0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.     

New generation pharmacogenomic tools: a SNP linkage disequilibrium Map,validated SNP assay resource,and high-throughput instrumentation system for large-scale genetic studies

Since public and private efforts announced the first draft of the human genome last year, researchers have reported great numbers of single nucleotide polymorphisms (SNPs). We believe that the availability of well-mapped, quality SNP markers constitutes the gateway to a revolution in genetics and personalized medicine that will lead to better diagnosis and treatment of common complex disorders. A new generation of tools and public SNP resources for pharmacogenomic and genetic studies--specifically for candidate-gene, candidate-region, and whole-genome association studies--will form part of the new scientific landscape. This will only be possible through the greater accessibility of SNP resources and superior high-throughput instrumentation-assay systems that enable affordable, highly productive large-scale genetic studies. We are contributing to this effort by developing a high-quality linkage disequilibrium SNP marker map and an accompanying set of ready-to-use, validated SNP assays across every gene in the human genome. This effort incorporates both the public sequence and SNP data sources, and Celera Genomics' human genome assembly and enormous resource ofphysically mapped SNPs (approximately 4,000,000 unique records). This article discusses our approach and methodology for designing the map, choosing quality SNPs, designing and validating these assays, and obtaining population frequency ofthe polymorphisms. We also discuss an advanced, high-performance SNP assay chemisty--a new generation of the TaqMan probe-based, 5' nuclease assay-and high-throughput instrumentation-software system for large-scale genotyping. We provide the new SNP map and validation information, validated SNP assays and reagents, and instrumentation systems as a novel resource for genetic discoveries.     

High-performance liquid chromatographic validated assay of doxorubicin in rat plasma and tissues

A specific and selective high-performance liquid chromatography (HPLC) technique that requires few manipulations, and is readily adaptable to analysis for a large series of samples, has been developed for the determination of the concentration of the anticancer drug doxorubicin (DXR) in rat serum and tissues. The biological samples were efficiently deproteinised and resolved from a reversed-phase nucleosil C₁₈ column with fluorescence detection. The validation study of the proposed method was successfully carried out in an assay range of between 5 and 5000 ng/ml and was subsequently implemented in a pharmacokinetic study of DXR in Wistar rats that were treated by intravenous administration of the drug.     

Fd的制备及其参与的HPLC—荧光法检测PaO活力研究

PaO体外活力测定需要Fd作还原剂。通过丙酮沉淀法、DEAE柱层析等制备菠菜Fd,并将其应用于HPLC—荧光法研究PaO催化的Pheide α降解反应。结果表明：经DEAE柱层析纯化得到的是氧化型Fd,而未经DEAE柱层析得到的是还原型Fd,还原型Fd可以参与Pheide α降解反应,色谱峰面积可以表示PaO的活力;FCC的HPLC洗脱时间是5．1min,遮光条件下FCC的半衰期是34．66min,PaO在20℃催化Pheide α降解的活力较高。小麦幼苗离体暗诱导衰老过程中PaO活力变化幅度较大,暗处理5d其活力增加24．47倍,“PaO”Chl降解途径在麦类作物叶片衰老过程中普遍存在。     

Strategic assay deployment as a method for countering analytical bottlenecks in high throughput process development: Case studies in ion exchange chromatography

High throughput approaches to facilitate the development of chromatographic separations have now been adopted widely in the biopharmaceutical industry, but issues of how to reduce the associated analytical burden remain. For example, acquiring experimental data by high level factorial designs in 96 well plates can place a considerable strain upon assay capabilities, generating a bottleneck that limits significantly the speed of process characterization. This article proposes an approach designed to counter this challenge; Strategic Assay Deployment (SAD). In SAD, a set of available analytical methods is investigated to determine which set of techniques is the most appropriate to use and how best to deploy these to reduce the consumption of analytical resources while still enabling accurate and complete process characterization. The approach is demonstrated by investigating how salt concentration and pH affect the binding of green fluorescent protein from Escherichia coli homogenate to an anion exchange resin presented in a 96‐well filter plate format. Compared with the deployment of routinely used analytical methods alone, the application of SAD reduced both the total assay time and total assay material consumption by at least 40% and 5%, respectively. SAD has significant utility in accelerating bioprocess development activities. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

False-negative rate and recovery efficiency performance of a validated sponge wipe sampling method

Recovery of spores from environmental surfaces varies due to sampling and analysis methods, spore size and characteristics, surface materials, and environmental conditions. Tests were performed to evaluate a new, validated sponge wipe method using Bacillus atrophaeus spores. Testing evaluated the effects of spore concentration and surface material on recovery efficiency (RE), false-negative rate (FNR), limit of detection (LOD), and their uncertainties. Ceramic tile and stainless steel had the highest mean RE values (48.9 and 48.1%, respectively). Faux leather, vinyl tile, and painted wood had mean RE values of 30.3, 25.6, and 25.5, respectively, while plastic had the lowest mean RE (9.8%). Results show roughly linear dependences of RE and FNR on surface roughness, with smoother surfaces resulting in higher mean REs and lower FNRs. REs were not influenced by the low spore concentrations tested (3.10 × 10(-3) to 1.86 CFU/cm(2)). Stainless steel had the lowest mean FNR (0.123), and plastic had the highest mean FNR (0.479). The LOD(90) (≥1 CFU detected 90% of the time) varied with surface material, from 0.015 CFU/cm(2) on stainless steel up to 0.039 on plastic. It may be possible to improve sampling results by considering surface roughness in selecting sampling locations and interpreting spore recovery data. Further, FNR values (calculated as a function of concentration and surface material) can be used presampling to calculate the numbers of samples for statistical sampling plans with desired performance and postsampling to calculate the confidence in characterization and clearance decisions.     

Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi.

The current problems with decreasing fossile resources and increasing environmental pollution by petrochemical-based plastics have stimulated investigations to find biosynthetic materials which are also biodegradable. Bacterial reserve materials such as polyhydroxyalkanoates (PHA) have been discovered to possess thermoplastic properties and can be synthesized from renewable resources. Poly-beta-hydroxybutyric acid (PHB) is at present the most promising PHA; and BIOPOL, its copolymer with poly-beta-hydroxy-valerate (PHV), is already industrially produced (ICI, UK), and used as packaging material (WELLA, FRG). According to the literature, PHA degradation has so far mainly been observed in bacteria; only under certain environmental conditions has fungal degradation of PHAs been indicated. Since fungi constitute an important part of microbial populations participating in degradation processes, a simple screening method for fungal degradation of BIOPOL, a PHA-based plastic, was developed. Several media with about 150 fungal strains from different terrestrial environments and belonging to different systematic and ecological groups were used. PHA depolymerization was tested on three PHB-based media, each with 0.1% BIOPOL or PHB homopolymer causing turbidity of the medium. The media contained either a comparatively low or high content of organic carbon (beside PHA) or were based on mineral medium with PHA as the principal source of carbon. The degradation activity was detectable due to formation of a clear halo around the colony (Petri plates) or a clear zone under the colony (test tubes).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interferences by anti-TB drugs in a validated HPLC assay for urinary catecholamines and their successful removal

A validated high performance liquid chromatographic assay for urinary catecholamines is presented. After addition of 3,4-dihydroxybenzylamine as internal standard (IS) to urine, norepinephrine (NE), epinephrine (E), dopamine (DA) are extracted by ion exchange chromatography and eluted with boric acid. After paired ion separation, quantitation is by electrochemical (coulometric) detection after correction of internal standard recovery. Novel interferences by anti-TB drugs on norepinephrine assay are discussed. A simple method for their removal using alumina is presented.     

The development of a microimmunoadsorbent assay and a microdialysis method for the evaluation of immunoadsorbent efficiency and antibody quantitation

A microimmunoadsorbent assay which provides for the quantitative elution and recovery of active antibody from immunoaffinity columns has been developed. We have also designed a microdialysis chamber which permits the dialysis of sample volumes from 50 to 500 microliters and allows quantitative recovery of macromolecular compounds. These techniques permit the rapid evaluation of a large number of eluants and immunoadsorbents in a short period of time. The microimmunoadsorbent assay coupled with the microdialysis method has been used to evaluate the efficacy of different anti-isopentenyl adenosine immunoadsorbents under various elution conditions. In the present study the best eluants for immunoaffinity purification of anti-N6-(delta 2-isopentenyl)adenosine antibody contained 15% pyridine in a sodium phosphate buffer or 4 M imidazole-HCl at pH 10.0.     

The development of a continuous isothermal titration calorimetric method for equilibrium studies

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12-20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60-180 min to generate 20-30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba(2+) to 18-crown-6 and the binding of cytidine 2'-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.     

A validated method for rapid analysis of ethane in breath and its application in kinetic studies in human volunteers

Oxidative stress may initiate lipid peroxidation that generates ethane. Ethane, at low concentrations, is eliminated by pulmonary exhalation. Previous methods have not allowed frequent sampling, thus ethane kinetics has not been studied in man. A validated method over the range 3.8-100,000 ppb with a limit of quantitation of 3.8 ppb (CV 9.3%) based on cryofocusing technique of a 60 ml breath sample allowed frequent sampling. Due to a rapid analytical procedure batches of more than 100 samples may be analyzed. In human volunteers (24-55 years) uptake was studied for up to 23 min (n=9), elimination was studied for 210 min (n=9). Ethane was inhaled (concentrations varied from 16 to 29 ppm (parts per million)) through a non-rebreathing system; sampling was performed with short intervals from the expiratory limb. Samples were also drawn from the inhalatory limb. Ninety-five percent of steady state (inspired) concentration was reached within 1.75 min. Five percent of the initially inhaled concentrations was found in exhaled air 1.5 min after termination of inhalation. A terminal mean half life of 31 min for ethane was also observed. The data indicate that frequent sampling will be necessary to capture relevant changes in breath ethane.     

Application of a validated high-performance liquid chromatography-mass spectrometry assay to the analysis of m- and p-hydroxybenzoylecgonine in meconium

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) assay, already validated for opiates and cocaine in meconium, has been re-applied for determination of m- and p-hydroxybenzoylecgonine, using nalorphine as the internal standard. Methodology included an initial extraction from the matrix by methanol and then a solid-phase extraction (SPE). A reversed-phase chromatography was used with a gradient of 1% acetic acid-acetonitrile coupled to atmospheric pressure ionization electrospray-mass spectrometry single ion monitoring mode. This method, validated in the range 0.005-1.00 microg analytes/g meconium, proved useful to identify and quantify these two metabolites in meconium samples, already tested for the presence of cocaine, benzoylecgonine and cocaethylene. A positivity of range of concentrations varied between 0.007 and 0.338 microg/g, confirming the importance of these two hydroxylated derivatives to monitor fetal exposure to cocaine.     

An easy-to-handle semi-automated method for media development using a colorimetric viability assay and fractional factorial designs

A rapid and effective semi-automated screening method has been developed for the development of growth media for mammalian cell culture. The method has proven to be a powerful tool for preliminary evaluation and comparison of new media formulations, but has some inherent disadvantages, which are important to recognise in order to interpret the results.     

Development and validation of a stability-indicating high performance liquid chromatographic (HPLC) assay for biperiden in bulk form and pharmaceutical dosage forms

Current compendial (USP) methods of assay for the analysis of biperiden in bulk form and pharmaceutical dosage forms involve the use of titrimetric and spectrophotometric procedures, respectively. These are non-selective and non-stability-indicating techniques. In this work, a stability-indicating high performance liquid chromatographic assay procedure has been developed and validated for biperiden. The liquid chromatographic separation was achieved isocratically on a symmetry C8 column (150 mm x 3.9 mm i.d., 5 microm particle size) using a mobile phase containing methanol-buffer (50:50, v/v, pH 2.50) at a flow rate of 1 ml/min and UV detection at 205 nm. The buffer was composed of sodium dihydrogen phosphate (50 mM) and 1-heptanesulfonic acid sodium salt (5 mM). The method was linear over the concentration range of 0.5-25 microg/ml (r=0.9998) with a limit of detection and quantitation 0.03 and 0.1 microg/ml, respectively. The method has the requisite accuracy, selectivity, sensitivity and precision to assay biperiden in bulk form and pharmaceutical dosage forms. Degradation products resulting from the stress studies did not interfere with the detection of biperiden and the assay is thus stability-indicating.