Simple fluorescence assay for quantification of OSCS in heparin

In 2008, heparin contaminated with oversulfated chondroitin sulfate (OSCS) penetrated the worldwide market and was associated with severe adverse effects. Feasible and reliable methods to test heparin for adulteration are needed. The objective was to develop a simple approach based on a microplate assay for quantification of heparin and sulfated glycans using the fluorescent heparin sensor polymer-H (polymer-H assay). However, both heparin and OSCS concentration-dependently increase the fluorescence intensity (FI) of polymer-H, so that OSCS in heparin cannot be detected. The idea was a two-step procedure including, first, separation of heparin by degradation with heparinase I, and then measurement of the remaining OSCS. To achieve complete heparin (unfractionated heparin (UFH), enoxaparin) degradation, several conditions (e.g. incubation time and heparinase I concentration) were optimized by using the aXa assay for monitoring. Defined UFH/OSCS mixtures incubated in this way showed a concentration-dependent FI increase in the polymer-H assay (λ _(em) 330 nm, λ _(ex) 510 nm). The sensitivity was unexpectedly high with an LOD/LOQ of 0.5%/0.6% OSCS content in heparin. Further experiments testing UFH/OSCS mixtures in the aXa assay confirmed our hypothesis: OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. In contrast with ¹H NMR and CE, neither expensive equipment nor much experience are required. It could be applied not only in the quality control of heparin, but also in clinical practice, to check the applied heparin preparation when a patient suffers any adverse effect.     

Determination of oversulfated chondroitin sulfate and dermatan sulfate impurities in heparin by capillary electrophoresis

Recently, oversulfated chondroitin sulfate (OSCS) present in certain lots of heparin was identified as the toxic contaminant responsible for severe side effects following intravenous heparin administration. The United States Pharmacopeia (USP) and European Pharmacopeia (Eur.Ph.) announced an immediate revision of their monographs for heparin sodium by adding two US Food and Drugs Administration-recommended tests for OSCS based on nuclear magnetic resonance and capillary electrophoresis (CE). However, the proposed CE method provides only partial separation of the OSCS contaminant from heparin, thereby hindering appropriate impurity profiling. Here we present an improved CE method that is especially useful for the reliable quantification of OSCS in heparin samples, but also allows determination of the common impurity dermatan sulfate (DS). Parameters such as type and concentration of background electrolyte, capillary temperature, sample concentration and injection volume were investigated and optimized. Enhancement of the OSCS–heparin separation was achieved by using high concentrations of Tris phosphate (pH 3.0) as background electrolyte. High currents and excessive Joule heating were prevented by employing fused-silica capillaries with an internal diameter of 25 μm. Good separations of OSCS, heparin and DS are obtained within 17 min. The method permits injection of relatively high heparin concentrations (up to 50 mg/ml) and large sample volumes (up to 5% of the capillary volume) allowing OSCS and DS determination in heparin down to the 0.05% and 0.5% (w/w) level, respectively. The CE method is shown to be repeatable and linear (R² > 0.99) for OSCS, heparin and DS. CE analyses of OSCS-contaminated heparin samples and different heparin standards further demonstrate the utility of the method.     

Characterization of currently marketed heparin products: key tests for quality assurance

During the 2007–2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500 MHz ¹H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007–2008 crisis.     

Capillary electrophoresis of heparin and other glycosaminoglycans using a polyamine running electrolyte

This study involves the use of polyamines as potential resolving agents for the capillary electrophoresis (CE) of glycosaminoglycans (GAGs), specifically heparin, dermatan sulfate, chondroitin sulfate, over-sulfated chondroitin sulfate (OSCS), and hyaluronan. All of the compounds can be separated from each other with the exception of chondroitin sulfate and hyaluronan. Using optimization software, the final run conditions are found to be 200 mM ethylenediamine and 45.5 mM phosphate as the electrolyte with −14 V applied across a 50 μm ID × 24.5 cm fused silica capillary at 15 °C. The ion migration order, with OSCS as the last instead of the first peak, is in contrast to previous reports using either a high molarity TRIS or lithium phosphate run buffer with narrower bore capillaries. Total analysis time is 12. 5 min and the relative standard deviation of the heparin migration time is about 2.5% (n = 5). The interaction mechanism between selected polyamines and heparin is explored using conductivity measurements in addition to CE experiments to show that an ion-pairing mechanism is likely.     

Identification of heparin samples that contain impurities or contaminants by chemometric pattern recognition analysis of proton NMR spectral data

Chemometric analysis of a set of one-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectral data for heparin sodium active pharmaceutical ingredient (API) samples was employed to distinguish USP-grade heparin samples from those containing oversulfated chondroitin sulfate (OSCS) contaminant and/or unacceptable levels of dermatan sulfate (DS) impurity. Three chemometric pattern recognition approaches were implemented: classification and regression tree (CART), artificial neural network (ANN), and support vector machine (SVM). Heparin sodium samples from various manufacturers were analyzed in 2008 and 2009 by 1D (1)H NMR, strong anion-exchange high-performance liquid chromatography, and percent galactosamine in total hexosamine tests. Based on these data, the samples were divided into three groups: Heparin, DS ≤ 1.0% and OSCS = 0%; DS, DS > 1.0% and OSCS = 0%; and OSCS, OSCS > 0% with any content of DS. Three data sets corresponding to different chemical shift regions (1.95-2.20, 3.10-5.70, and 1.95-5.70 ppm) were evaluated. While all three chemometric approaches were able to effectively model the data in the 1.95-2.20 ppm region, SVM was found to substantially outperform CART and ANN for data in the 3.10-5.70 ppm region in terms of classification success rate. A 100% prediction rate was frequently achieved for discrimination between heparin and OSCS samples. The majority of classification errors between heparin and DS involved cases where the DS content was close to the 1.0% DS borderline between the two classes. When these borderline samples were removed, nearly perfect classification results were attained. Satisfactory results were achieved when the resulting models were challenged by test samples containing blends of heparin APIs spiked with non-, partially, or fully oversulfated chondroitin sulfate A, heparan sulfate, or DS at the 1.0%, 5.0%, and 10.0% (w/w) levels. This study demonstrated that the combination of 1D (1)H NMR spectroscopy with multivariate chemometric methods is a nonsubjective, statistics-based approach for heparin quality control and purity assessment that, once standardized, minimizes the need for expert analysts.     

问题肝素中多硫酸软骨素杂质的柱前衍生高效液相色谱分析

基于肝素和多硫酸软骨素(OSCS)在单糖组成上的差别,建立了可用于肝素中OSCS检测的柱前衍生高效液相色谱法.采用3 mol/L三氟乙酸,将受污染的问题肝素在110℃下充氮封管水解4 h,在碱性条件下与1-苯基-3-甲基-5-吡唑啉酮进行衍生化反应,再采用C18反相色谱柱,以0.1 mol/L磷酸盐(pH=6.7)缓冲液/乙腈(体积比82∶18)为流动相,在流速1.0 mL/min、柱温25℃及紫外检测波长245 nm的条件下进行液相色谱分析.结果表明,肝素和OSCS的单糖色谱峰具有良好的分离度,测得2批问题肝素中OSCS杂质的质量分数分别为19.6%和28.3%.该方法具有良好的精密度和重现性,易于推广,适合于肝素中OSCS杂质的检测,并可用于硫酸软骨素A和C与硫酸软骨素B的区分和鉴别.     

Purification and Characterization of a Novel Heparin Degrading Enzyme from <Emphasis Type="Italic">Aspergillus flavus</Emphasis> (MTCC-8654)

A heparinase-producing fungus was isolated, and the strain was taxonomically characterized as Aspergillus flavus by morphophysiological and 26S rRNA gene homology studies. The culture produced intracellular heparinase enzyme, which was purified 40.5-fold by DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatography. Specific activity of the purified enzyme was found to be 44.6 IU/μg protein and the molecular weight of native as well as reduced heparinase was 24 kDa, showing a monomeric unit structure. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. The enzyme activity was maximum at 30 °C in the presence of 300 mM NaCl at pH 7.0. In the presence of Co²⁺, Mn²⁺ ions, and reducing agents (β-mercaptoethanol, dithiothreitol), enzyme activity was enhanced and inhibited by iodoacetic acid. These observations suggested that free sulfohydryl groups of cysteine residues were necessary for catalytic activity of the enzyme. The enzyme was also inhibited by histidine modifier, DEPC, which suggests that along with cysteine, histidine may be present at its active site. The enzyme showed a high affinity for heparin as a substrate with K _m and V _max as 2.2 × 10⁻⁵ M and 30.8 mM min⁻¹, respectively. The affinity of the enzyme for different glycosaminoglycans studied varied, with high substrate specificity toward heparin and heparin-derived polysaccharides. Depolymerization of heparin and fractionation of the oligosaccharides yielded heparin disaccharides as main product.     

污染肝素中多硫酸化硫酸软骨素的分步醋酸纤维素薄膜电泳分析

采用离子交换色谱法从污染肝素原料中分离出多硫酸化硫酸软骨素(OSCS),建立了分步醋酸纤维素薄膜电泳法分析污染肝素中OSCS含量的方法.结果表明,先以0.05 mol/L醋酸钡缓冲液(pH 5.0)电泳,再以0.15 mol/L醋酸锌缓冲液(pH 6.3)电泳,可以将肝素和OSCS完全分开,检出限为0.1 g/L; 通过灰度积分建立定量校准曲线,相关系数为0.9934,平均回收率为102.1%～106.1%; RSD为4.1%～6.0%.     

Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II)

The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.     

Quantitative Determination of High Charge Density Polyanion Contaminants in Biomedical Heparin Preparations Using Potentiometric Polyanion Sensors

Quantification of oversulfated chondroitin sulfate (OSCS) in biomedical heparin preparations is achieved using a recently described potentiometric polyanion sensor-based approach operated in a kinetic mode of analysis. This is accomplished by adjusting the concentration of the test sample to a range where the OSCS level is low enough for the sensor not to achieve a full and rapid equilibrium phase boundary potential change at the membrane/sample interface upon exposure to the heparin sample. Using this method, the OSCS wt% determined within heparin samples containing OSCS are shown to be in good agreement with those determined by an accepted NMR method.     

Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-I digest patterns

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M _w) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A ₂) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M _w of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M _w values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M _w results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.     

A microfluidic system for evaluation of antioxidant capacity based on a peroxyoxalate chemiluminescence assay

A microfluidic system incorporating chemiluminescence detection is reported as a new tool for measuring antioxidant capacity. The detection is based on a peroxyoxalate chemiluminescence (PO-CL) assay with 9,10-bis-(phenylethynyl)anthracene (BPEA) as the fluorescent probe and hydrogen peroxide as the oxidant. Antioxidant plugs injected into the hydrogen peroxide stream result in inhibition of the CL emission which can be quantified and correlated with antioxidant capacity. The PO-CL assay is performed in 800-μm-wide and 800-μm-deep microchannels on a poly(dimethylsiloxane) (PDMS) microchip. Controlled injection of the antioxidant plugs is performed through an injection valve. Of the plant-food based antioxidants tested, β-carotene was found to be the most efficient hydrogen peroxide scavenger (SA _HP of 3.27 × 10⁻³ μmol⁻¹ L), followed by α-tocopherol (SA _HP of 2.36 × 10⁻³ μmol⁻¹ L) and quercetin (SA _HP of 0.31 × 10⁻³ μmol⁻¹ L). Although the method is inherently simple and rapid, excellent analytical performance is afforded in terms of sensitivity, dynamic range, and precision, with RSD values typically below 1.5%. We expect our microfluidic devices to be used for in-the-field antioxidant capacity screening of plant-sourced food and pharmaceutical supplements. Figure Assembled PDMS microchip sandwiched between two glass plates with the top plate containing capillary reservoirs     

An investigation of heparinase immobilization

A systematic investigation of the parameters that affect the efficiency of immobilizing heparinase onto cyanogen bromide activated crosslinked 8% agarose beads was conducted. Two experimental measures, the “fraction bound” and the “fraction retained,” were used to monitor the coupling efficiency. The fraction bound is the portion of the total initial enzyme that is bound to the agarose gel. The fraction retained is the fraction of bound enzyme that is active. The product of the two measures indicates the coupling efficiency. The activity of the immobilized heparinase was measured under conditions free of both internal and external mass transfer limitations, and thus, the fraction retained represents the true immobilized enzyme activity. Increasing the degree of activation of the beads results in an increase in the fraction bound, the fraction retained, and consequently, the coupling efficiency. As the ratio of enzyme solution to gel volume increases from 1.5 to 2.2, the fraction bound remains constant but the fraction retained decreases (heparinase concentration; 0.15 mg/mL and degree of activation; 9.5 μmol of cyanate esters/g of gel). At volume ratios greater than 2.2, both the fraction bound and the fraction retained decline continuously. Changing the heparinase concentration in the coupling solution changes the coupling efficiency in a manner similar to that of the volume ratio change. When heparin is added during the coupling process, the fraction bound declines as the heparin concentration increases, whereas the fraction retained increases up to a heparin concentration of 12 mg/mL and decreases thereafter. When arginine, lysine, and glycine are used to block the unreacted cyanate ester groups after the coupling process, the immobilized heparinase shows different pH optima of 6.5, 6.9, and 7.2, respectively. Based upon these findings, a protocol to optimize heparinase immobilization is developed.     

Exploiting flow injection system with mini-immunoaffinity chromatographic column for chondroitin sulfate proteoglycans assay

A flow injection (FI) system with a mini-immunoaffinity chromatographic column was used to perform on-line assays of specific proteoglycans. The 300-μL mini-column contained beads coupled with monoclonal antibodies against the specific sulfation pattern of chondroitin sulfate proteoglycans, which have been reported to be a potential biomarker for cancer. The amount of these proteoglycans present was estimated indirectly from their protein content using the Bradford assay, which is an alternative to a direct carbohydrate assay. The system developed was tested by assaying for chondroitin sulfate proteoglycans in sera from patients with various cancers and comparing the results to those obtained for sera from healthy people. The results indicated that this approach could be used as a cost-effective alternative system for determining the amount of these specific biomarker proteoglycans. The column could be reused at least 90 times, with each run consisting of 200 μL of serum sample diluted twofold; an analysis rate of 30 min per run was achieved, as compared to 4 h for a batch procedure.     

High-sensitivity visualisation of contaminants in heparin samples by spectral filtering of 1H NMR spectra

A novel application of two-dimensional correlation analysis has been employed to filter (1)H NMR heparin spectra distinguishing acceptable natural variation and the presence of foreign species. Analysis of contaminated heparin samples, compared to a dataset of accepted heparin samples using two-dimensional correlation spectroscopic analysis of their 1-dimensional (1)H NMR spectra, allowed the spectral features of contaminants to be recovered with high sensitivity, without having to resort to more complicated NMR experiments. Contaminants, which exhibited features distinct from those of heparin and those with features normally hidden within the spectral mass of heparin could be distinguished readily. A heparin sample which had been pre-mixed with a known contaminant, oversulfated chondroitin sulfate (OSCS), was tested against the heparin reference library. It was possible to recover the (1)H NMR spectrum of the OSCS component through difference 2D-COS power spectrum analysis of as little as 0.25% (w/w) with ease, and of 2% (w/w) for more challenging contaminants, whose NMR signals fell under those of heparin. The approach shows great promise for the quality control of heparin and provides the basis for greatly improved regulatory control for the analysis of heparin, as well as other intrinsically heterogeneous and varied products.     

Flow and Sequential Injection Manifolds for the Spectrophotometric Determination of Captopril Based on its Oxidation by Fe(III)

 Two new simple and rapid methods are reported for the accurate and precise spectrophotometric determination of captopril (CPL) using flow (FI) and sequential injection (SI) analysis. The methods are based on the fast oxidation of CPL by Fe(III). The produced Fe(II) reacts with 2,2′-dipyridyl-2-pyridylhydrazone (DPPH) in acidic medium to form a colored complex which is monitored spectrophotometrically at 535 nm. Both methods allow the determination of the analyte up to 1000 mg L⁻¹ at a sampling rate of 120 and 60 injections per hour for FI and SI, respectively. The methods are very precise [s _r=0.8 and 1.2% at 500 mg L⁻¹ CPL (n=12) for FI and SI, respectively] and the 3σ detection limits (c _L=4.0 and 7.0 mg L¹, respectively) are quite satisfactory. Their application to a variety of anti-hypertensive commercial pharmaceutical formulations showed excellent results (relative errors, e _r, < ± 1.6% in all cases compared to an official HPLC method), while common pharmaceutical excipients were found not to interfere. Recovery experiments further verified the accuracy of the developed methods, as the percent recoveries were in the range of 98.1–102.5%. Author for correspondence. E-mail: themelis@chem.auth.gr Received May 9, 2002; accepted January 8, 2003 Published online May 5, 2003     

Development of a monoclonal antibody-based, congener-specific and solvent-tolerable direct enzyme-linked immunosorbent assay for the detection of 2,2',4,4'-tetrabromodiphenyl ether in environmental samples

A sensitive direct enzyme-linked immunosorbent assay (ELISA) for the specific detection of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in environmental samples was developed. A hapten mimicking BDE-47 was synthesized by introducing a butyric acid spacer into 5-hydroxy-BDE-47 and coupled to keyhole limpet hemocyanin to form an immunogen for the production of monoclonal antibodies (Mabs) against BDE-47. The most sensitive direct ELISA was formatted with a Mab, designated as 4F2, in combination with 5-(2,4-dibromophenoxy)pentanoic acid peroxidase as a tracer. The inhibition half-maximum concentrations and limit of detection of BDE-47 in phosphate buffered saline with 25% DMSO were 1.4 ± 0.05 and 0.1 ng mL⁻¹, respectively. Cross-reactivity values of the ELISA with the tested BDE congeners and metabolites were ≤5.8%. This assay was used to determine BDE-47 in soil, sediment and house dust samples after ultrasonic extraction, simple cleanup and concentration steps. The average recoveries, repeatabilities (intraday extractions and analyses), and intra-laboratory reproducibilities (interday extractions and analyses) were in a range of 92–126%, 8–19% and 9–25%, respectively. Applied to 44 real samples, the results of this assay displayed a statistically significant correlation with those of a gas chromatography–mass spectrometry method (R ² = 0.79-0.85), indicating this ELISA is a suitable tool for environmental analyses of BDE-47.     

Analysis and characterization of heparin impurities

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007?C2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations.

New noncellular fluorescence microplate screening assay for scavenging activity against singlet oxygen

In the present study, a new fluorescence microplate screening assay for evaluating scavenging activity against singlet oxygen (¹O₂) was implemented. The chemical generation of ¹O₂ was promoted using the thermodissociable endoperoxide of disodium 3,3′-(1,4-naphthalene)bispropionate (NDPO₂). The detection of ¹O₂ was achieved using dihydrorhodamine 123 (DHR), a nonfluorescent molecule that is oxidizable to the fluorescent form rhodamine 123 (RH). The combined use of a ¹O₂-selective generator and a highly sensitive probe (DHR) was then successfully applied to perform a screening assay of the ¹O₂ scavenging activities of ascorbic acid, penicillamine, cysteine, N-acetylcysteine (NAC), methionine, reduced glutathione (GSH), dihydrolipoic acid, lipoic acid, and sodium azide. All of these antioxidants exhibited concentration-dependent ¹O₂ scavenging capacities. They could be ranked according to observed activity: ascorbic acid> cysteine> penicillamine> dihydrolipoic acid > GSH> NAC> sodium azide> lipoic acid (IC₅₀ values of 3.0 ± 0.2, 8.0 ± 0.7, 10.9 ± 0.8, 25.2 ± 4.5, 57.4 ± 5.9, 138 ± 13, 1124 ± 128, 2775 ± 359 μM, mean±SEM, respectively) > methionine (35% of scavenging effect at 10 mM). In conclusion, the use of NDPO₂ as a selective generator for ¹O₂ and its fluorescence detection by the highly sensitive probe DHR is shown to be a reliable and resourceful analytical alternative means to implement a microplate screening assay for scavenging activity against ¹O₂. Generation and detection of singlet oxygen     

Method Development and Validation of a Rapid Determination of Ropinirole in Tablets by LC-UV

A reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed and validated for the determination of ropinirole (ROP) in tablets. The assay utilized UV detection at 250 nm and a Luna CN column (250 × 4.6 mm I.D, 5 μm). The mobile phases were comprised of acetonitrile: 10 mM nitric acid (pH 3.0) (75:25, v/v). Validation experiments were performed to demonstrate linearity, accuracy, precision, limit of quantitation (LOQ), limit of detection (LOD), and robustness. The method was linear over the concentration range of 0.5–10.0 μg mL⁻¹. The method showed good recoveries (99.75–100.20%) and the relative standard deviations of intra and inter-day assays were 0.38–1.69 and 0.45–1.95%, respectively. The method can be used for quality control assay of ropinirole.