Flavonoid composition of orange peel and its association with antioxidant and anti-inflammatory activities.

Dried citrus peel derived from Citrus reticulata, also called "chenpi", possesses a complex mixture of flavonoids and has a history of traditional use to treat a variety of digestive disorders. We compared three sources of conventional chenpi from California (USA), Guangxi, Zhejiang, and two sources of "nchenpi", which contain greater nobiletin content, from Sichuan and Xinhui (China). Xinhui orange peel extract (OPE) had highest content of polymethoxylated flavones, along with greatest capacity to scavenge 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-pcrylhydrazyl (DPPH), and 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) radicals and nitric oxide (NO). OPE also had higher NO, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) inhibitory activity than an equivalent mixture of flavonoids (P<0.05). In conclusion, nobiletin is a good chemical marker for assessing the anti-inflammatory potential of OPE from different sources. Obtaining "nchenpi" from either Sichuan or Xinhui provided potentially superior health benefits compared to conventional chenpi sources.

Avaliação fenotípica in vitro de novas amidinas aromáticas sobre Trypanosoma cruzi / Phenotypic screening in vitro of novel aromatic amidines on trypanosoma cruzi

A doença de Chagas (DC), causada pelo protozoário Trypanosoma cruzi, é uma doença negligenciada endêmica em diferentes regiões empobrecidas da América Latina. O tratamento, baseado em dois nitroderivados, Nifurtimox e Benzonidazol(Bz), é insatisfatório, demandando a busca de novos fármacos com ação tripanocida que sejam mais seletivos e eficazes. Nesse âmbito, o presente trabalho busca a identificação de novos agentes antiparasitários para a DC, explorando a avaliação fenotípica de novas amidinas aromáticas sintéticas in vitro incluindo ensaios de combinação entre estes compostos. Dez novas amidinas foram testadas sobre tripomastigotas sanguíneos e amastigotas de diferentes cepas do T. cruzi (Y eTulahuen) e também sobre células de mamífero hospedeiras (linhagem L929 ecélulas cardíacas) para determinar seu perfil eficácia e de toxicidade,respectivamente. Dentre as moléculas testadas (apresentando um ou dois grupamentos catiônicos terminais), cinco foram mais ativas sobre tripomastigotas sanguíneos que o fármaco de referência (Bz), sendo uma delas, a DB2267(molécula dicatiônica) a mais eficaz, exibindo EC50 de 0,23 miM e um índice de seletividade (IS) de 417. Esta diamidina foi 28 vezes mais ativa e cerca de três vezes mais seletiva que Bz. Para determinar se a combinação de duas amidinas teria um efeito tripanocida superior ao seu uso em monoterapia, tripomastigotas sanguíneos foram incubados com DB2267 e DB2236 em proporções fixas e os resultados mostraram apenas um efeito aditivo com sigmaFIC<4. Interessantemente,quando formas intracelulares foram expostas à DB2267, sua atividade foi relacionada à cepa do parasito, sendo eficaz (EC50 = 0.87 ± 0.05 miM) contra DTU II(cepa Y), mas não contra um representante da DTU VI (Tulahuen), mesmo quando utilizamos veículo diferente do DMSO (beta-ciclodextrina)...

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a neglecteddisease endemic in different poor areas of Latin America. The treatment, based ontwo nitroderivatives, Nifurtimox and Benznidazole (Bz), is unsatisfactory, demandingthe screening of new potential trypanocidal drugs more selective and potent. In thisscope, the present work deals with the identification of new anti-parasitic agents forCD, exploring the phenotypic screening of novel synthetic aromatic amidines in vitroand also combination assays between these compounds. The novel ten amidineswere tested against bloodstream trypomastigotes (BT) and amastigote forms ofdifferent T.cruzi strains (Y and Tulahuen) and were also evaluated on mammalianhost cells (L929 cells and cardiac cells) to check their toxicity profile. Among thesemolecules, five were more active against BT than the reference drug (Bz), being oneof them, the DB2267 (a dicationic molecule) the most potent, exhibiting an EC50value of 0.23 µM and a selectivity index (SI) of 417. This diamidine was 28-fold moreactive and about 3 times more selective than Bz. To ascertain if the combination oftwo amidines could improve their trypanocidal activity, BT were incubated withDB2267 and DB2236 in fixed-ratio proportions and the data showed only an additiveeffect with sigmaFIC<4. Interestingly, when intracellular forms were exposed to DB2267,its activity was related to the parasite strain, being effective (EC50 = 0.87 ± 0.05 miM)against DTU II (Y strain) but not against one representative of DTU VI (Tulahuen)even using different vehicles (beta-cyclodextrins and DMSO)...

Determination of the thrombin inhibitor AZD0837 and its metabolites in human bile using mixed mode solid phase extraction and LC-MS/MS.

A method for the determination of AZD0837 and its two metabolites AR-H069927 and AR-H067637 in human bile was developed and validated. All three analytes and their stable isotope-labeled internal standards were isolated from bile using solid phase extraction on a mixed mode reversed phase/anion exchange column. Elution was done at high ionic strength with 0.125 M ammoniumacetate in 50% methanol. The extraction recoveries were >75%. Due to the high concentration of AR-H067637 a portion of the extract was diluted before injection on to the LC column, while undiluted extract was directly injected for the analysis of AZD0837 and AR-H069927. Chromatographic separation of all three analytes was achieved in a single system utilizing a C18 column based on fused core particle technology at high flow rate. The two metabolites were eluted when a gradient from 30 to 57% methanol was applied while the more hydrophobic pro-drug, AZD0837, eluted during a steeper second gradient from 57 to 80% methanol with the ammonium acetate concentration and acetic acid concentration kept constant at 3.8 mmol/L and 0.1%, respectively. The total cycle time was 3.2 min. Detection was performed using positive electrospray ionization tandem mass spectrometry. The linearity range was 0.02-20 µmol/L for AZD0837 and AR-H069927, and 1-1000 µmol/L for AR-H067637. The repeatability and the overall precision were less than 15% (RSD) and the accuracy was within the interval 93-100%.

[AAPH scavenging activities of 22 flavonoids and phenolic acids and 9 extracts of Chinese materia medica].

OBJECTIVE: To investigate the AAPH scavenging activities of 22 flavonoids and phenolic acids and 9 extracts of Chinese materia medica. METHOD: The antioxidant activities of the samples were evaluated by an oxygen radical absorbance capacity method (ORAC), at the same time, the total contents of flavonoids and phenolic the 9 herb extracts were analyzed by Folin-Ciocalteu method, and the active components were qualitatively and quantitatively analyzed by an HPLC method. RESULT: It was found that the tea extract showed the strongest AAPH activity with the ORAC value of 4786.40 micromol x g(-1) whereas safflower demonstrated the weakest activity with the ORAC value of 784.04 micromol x g(-1). As for compounds, quercetin had the strongest AAPH activity with the ORAC value of 12.90 while ( - )-EGC had the weakest activity with the ORAC value of 2.47. A quantitative relationship was obtained to describe the AAPH scavenging activity of the herb extracts: Y = 1844.8 lnX-3577.5, r = 0.8675, where Y stands for the ORAC vaule, and X stands for the concentration of total phenolic acids. CONCLUSION: Flavonoids and phenolic acids are the AAPH scavenging active ingredients in the Chinese herb extracts. It's a good way to study the antioxidant activity of Chinese herb extract and its chemical composition by combing ORAC method and HPLC method.

Efficiency evaluation of the main multiresidue methods used in Europe for the analysis of amitraz and its major metabolites.

This paper compares the performance of the three most widely employed multiresidue methods [quick, easy, cheap, effective, rugged, and safe (QuEChERS), mini-Luke, and ethyl acetate] currently used for the determination of amitraz residues in fruits. A fast and differentiated analysis of amitraz and its two main metabolites, N-2,4-dimethylphenyl-N-methylformamidine and 2,4-dimethylformanilide, was performed by HPLC-electrospray ionization-MS/MS using a triple quadrupole mass spectrometer in the positive mode. A test of the stability of the standard solutions showed a rapid hydrolysis of amitraz to the amide and amidine derivatives in solutions containing water, including QuEChERS extracts of crops that were previously acidified. Two useful mass transitions were used to confirm the presence of each analyte in the sample extracts. LOD values ranging from 0.4 to 2.0 microglkg were obtained. Linearity of response over 2 orders of magnitude was demonstrated (r2 > 0.999) in solvent and pear extract. The recovery studies were performed on pear blanks spiked at two concentration levels, 50 and 500 microg/kg (n = 5). Best recoveries, ranging from 75 to 103%, were obtained by the application of the QuEChERS method with CV < 8% in all cases. The QuEChERS method was applied to a monitoring study carried out by the Chemical and Veterinary Investigation Office Stuttgart laboratory. From the 63 pear samples analyzed, 21 contained amitraz residues (expressed as sum) ranging from 0.02 to 2.9 mg/kg. Amitraz parent was detected only in a few cases at very low concentration levels, with N-2,4-dimethylphenyl-N-methylformamidine being the metabolite almost entirely representing the total residue. These results emphasize that the residue situation is clearly underestimated if only the parent compound is targeted, and they reinforce how important it is to include amitraz in the target scope of pesticide residue laboratories, especially since the concentrations detected exceeded the Acute Reference Dose in the majority of cases and pose a health risk to the consumer.

FT-IR and theoretical study of 3,5-dimethyl-1H-pyrazole-1-carboxamidine (L) and the complexes CoL2(H2O)2(NO3)2, NiL2(H2O)2(NO3)2.

In the paper a joint experimental and theoretical study of 3,5-dimethyl-1H-pyrazole-1-carboxamidine (L) as well as its complexes CoL2(H2O)2(NO3)2 and NiL2(H2O)2(NO3)2 is reported. On the basis of FT-IR experiments and a DFT-derived scaled quantum mechanical force field the normal coordinate analysis of L was carried out. The FT-IR spectra of the two complexes were interpreted using the present assignment of L and computed vibrational data of the complexes. The ionic and charge transfer interactions in the complexes were assessed by means of natural bond orbital (NBO) analysis.

Validation and implementation of drug-dependent antibody assays in clinical trials for safety monitoring of patients dosed with roxifiban, an orally bioavailable glycoprotein IIb/IIIa antagonist.

Thrombocytopenia exposes patients to increased bleeding risk. This serious adverse event was observed with a frequency of approximately 2% in early clinical trials with the potent, orally bioavailable glycoprotein (GP) IIb/IIIa receptor antagonist roxifiban. We previously reported that drug-dependent antibodies (DDAbs) to GP IIb/IIIa are the main cause of thrombocytopenia with roxifiban. Two ELISA assays for detection of free DDAbs (in citrate plasma) and total DDAbs (in EDTA plasma to elute platelet bound DDAbs) were developed and analytically validated. These tests served two purposes during the clinical development program, to pre-screen patients for pre-existing antibodies and monitor patients for increasing antibody titers as a surrogate for eminent thrombocytopenia. The free DDAb assay showed inter and intra-assay precision of 5-12 and 12-14% CV, respectively. The total DDAb assay showed a precision of 5-10 and 4-12% CV, respectively. Three cycles of freeze-thaw did not significantly alter DDAb values in citrate plasma, EDTA plasma or extraction solution. The clinical qualifications of the two assays were conducted in two phase II clinical trials in coronary arterial disease (CAD) patients dosed with roxifiban. Both assays have demonstrated clinical sensitivity of nearly 99-100% and clinical specificity of nearly 95%.

Identification of pesticide transformation products in food by liquid chromatography/time-of-flight mass spectrometry via "fragmentation-degradation" relationships.

The identification of transformation products of pesticides in foodstuffs is a crucial task difficult to tackle, due to the lack of standards and scarce information available. In this work, we describe a methodology for the identification and structural elucidation of pesticide transformation products in food. The proposed strategy is based on the use of liquid chromatography electrospray time-of-flight mass spectrometry (LC/TOFMS): accurate mass measurements of (molecule and fragment) ions of interest are used in order to establish relationships between fragmentation of the parent pesticides in the instrument (in-source CID fragmentation) and possible degradation products of these pesticides in food. Examples of this strategy showing the potential of LC/TOFMS to determine unknown pesticides in food are described in two different real samples, suggesting that pesticides often are transformed into degradation products in the same fashion that they are fragmented in the instrument. Using the proposed approach and without using standards a priori, based solely on accurate mass measurements of ions and "fragmentation-degradation" relationships, we have identified two parent pesticides (amitraz and malathion) along with six degradation products, m/z 253 (N,N'-bisdimethylphenylformamidine), 163 (N-2,4-dimethylphenyl-N-methyl formamidine), 150 (2,4-dimethylformamidine), and 122 (2,4-dimethylaniline) from amitraz, and m/z 317 and 303, due to ether hydrolysis of methyl and ethyl groups from malathion. Structures for these species were proposed, and the potential of the proposed approach was critically discussed.

Radiosynthesis of (E)-N-(2-[11C]methoxybenzyl)-3-phenyl-acrylamidine, a novel subnanomolar NR2B subtype-selective NMDA receptor antagonist.

Recently, a novel series of amidines has been described, exhibiting high NR2B-subtype selective N-methyl-D-aspartate (NMDA) antagonist activity with nanomolar or subnanomolar affinity. Within the styrylamidine subclass, (E)-N-(2-methoxybenzyl)-3-phenyl-acrylamidine (1), displayed the highest affinity (Ki=0.7 nM versus [(3)H]ifenprodil) and was considered an appropriate candidate for isotopic labelling with carbon-11 (T(1/2): 20.38 min) at its methoxy group for imaging of NMDA receptors with PET. Derivative 1 has been labelled from the corresponding nor-analogue using [(11)C]methyl triflate and the following experimental conditions : (1) trapping at -10 degrees C of [(11)C]methyl triflate in 300 microL of acetone containing 0.6-0.8 mg of precursor 5 (2.4-3.2 micromol) and 5 microL of a 3M solution of NaOH in water (about 5 eq.); (2) concentration to dryness of the reaction mixture (at 110 degrees C, using a helium stream for 1-2 min); (3) taking up the residue with 0.5 mL of the HPLC mobile phase and (4) purification using semi-preparative HPLC (SymmetryPrep) C-18, Waters, 300 x 7.8 mm). Typically, starting from a 1.5 Ci (55.5 GBq) [(11)C]CO(2) production batch, 120-240 m Ci (4.44-8.88 GBq) of [(11)C]-1 (20-40% decay-corrected radiochemical yield, n=5) was obtained within a total synthesis time of 25-30 min. Specific radioactivities ranged from 0.8 to 1.2 Ci/micromol (29.6-44.4 GBq/micromol) at the end of radiosynthesis. No attempts were made to further optimise these reactions, as sufficient material was obtained to allow for preliminary pharmacological characterisation.

Formation of alpha-aminoadipic and gamma-glutamic semialdehydes in proteins by the maillard reaction.

Recent research has demonstrated that nonenzymatic glycation (the Maillard reaction) lead to the formation of carbonyl groups and advanced glycation end products (AGEs) in proteins. Such oxidative modifications are a major contributing factor to diabetic complications and aging. alpha-Aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) have been identified as the major carbonyl products in oxidized proteins both in vitro and in vivo. AAS is an oxidative deamination product of lysine residue, while GGS originates from arginine and proline residues. To evaluate oxidative damage to proteins by the Maillard reaction, we developed a method of detecting AAS and GGS by high-performance liquid chromatography (HPLC). The aldehydic residues in proteins were derivatized by reductive amination with NaCNBH3 and p-aminobenzoic acid (ABA), a fluorescence regent. After acid hydrolysis of the ABA-derivatized protein, ABA-AAS and ABA-GGS were measured by fluorometric HPLC. Thus, AAS and GGS could be detected in various proteins such as human plasma protein using the present method. Accumulation of both aldehydic residues was observed in oxidized proteins by reactive oxygen species. Furthermore, AAS and GGS were markedly formed in the incubation of BSA with ascorbic acid. The formation of both aldehydic residues was also observed in the incubation of BSA with 100 mM glucose or 1.0 mM methylglyoxal in the absence and presence of 100 microM Fe3+ for 2 weeks. These results suggest that the Maillard reaction can contribute to the formation of AAS and GGS in vivo.

Environmental impacts of diesel fuel on bacteria and phytoplankton in a tropical estuary assessed using in situ mesocosms.

Dissolved or dispersed petroleum hydrocarbon concentrations (DDPH) were monitored in Ponggol estuary, Singapore, fortnightly from July 1999 to June 2000. DDPH concentrations ranged from 4.4 to 248.9 microg l(-1) and 0.4 to 1099.7 microg l(-1) for surface and subsurface waters, respectively and with mean concentrations of 41.01 microg l(-1) in the water column. Absorbed or adsorbed petroleum hydrocarbon (AAPH) concentrations measured in sediments ranged from 20.6 to 541.0 mg kg(-1), with mean concentrations of 148.23 mg kg(-1). In situ mesocosm studies of bacteria and phytoplankton were based on field monitoring of environmentally measured concentrations of petroleum hydrocarbons, using diesel fuel as the source of contaminant. The mesocosm comprised of 25 L clear polycarbonate carboys incubated in situ for 6 days. Water and sediments from a clean site with undetectable levels of petroleum hydrocarbons were used in controls. The treatment mesocosms comprised of mean and highest concentrations of DDPH and AAPH. The study revealed signs of acute toxicity to autotrophs viz., phytoplankton and autotrophic bacteria in treatments simulating concentrations of diesel fuel found in the sediments. A stimulatory effect was seen at lower concentrations. Bacterial heterotrophs responded positively to all concentrations of diesel fuel because of the abundance of a carbon source, reduced grazing pressure and reduced competition for nutrients from phytoplankton.

Use of 2,2'-azobis(2-amidinopropane) dihydrochloride as a reagent tool for evaluation of oxidative stability of drugs.

PURPOSE: To study the oxidative degradation of drugs using a hydrophilic free radical initiator, 2,2'-Azobis(-amidinopropane) dihydrochloride (AAPH). METHODS: AAPH was used as the free radical initiator to study oxidation of three model compounds (A, B, and C), which represent different oxidizable moieties. In the solution model, the drugs and AAPH were dissolved in a mixture of acetonitrile and aqueous buffer and incubated at elevated temperatures to evaluate oxidative degradation. The effects of pH and drug-AAPH ratio on the kinetics of the reaction were evaluated for compound A. Commonly used antioxidants were also evaluated by addition to solutions of drug and AAPH. In the solid-state model, blends of drug with microcrystalline cellulose were treated with AAPH and placed at elevated temperature and humidity to evaluate solid state oxidation. RESULTS: Use of AAPH resulted in selective oxidation of the model drugs by a free radical initiated process. The scope of the technique was further investigated in detail using compound A. The rate of oxidation of compound A varied directly with the concentration of AAPH. The pseudo first-order rate constants for the oxidative degradation were calculated from the kinetic data. The antioxidants were rank-ordered based on their quenching activity on the rates of AAPH initiated oxidation for compound A. The concept was extended to oxidation in solid state. CONCLUSIONS: The proposed AAPH model is useful in assessing oxidative stability of drug candidates in development.

ESR study of a biological assay on whole blood: antioxidant efficiency of various vitamins.

This study deals with the activity of various vitamins against the radical-mediated oxidative damage in human whole blood. We have used a biological method that allows both the evaluation of plasma and that of red blood cell resistance against the free radicals induced by 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). Spin trapping measures using mainly 5-(diethoxyphosphoryl)-5-methyl-1-pyrolline N-oxide nitrone (DEPMPO) were carried out under several conditions to identify the free radicals implicated in this test. Only the oxygenated-centred radical generated from AAPH was found highly reactive to initiate red blood cell lysis. With DEPMPO only alkoxyl radicals were observed and no evidence was found for alkylperoxyl radicals. The antioxidant activity of several lipid- and water-soluble vitamins has been assessed by the biological assay and through two chemical methods. We have noticed high antioxidant activities for tocopherols (in the order delta>gamma>alpha) in the biological test but not through chemical methods. At 1 microM, the delta-tocopherol efficiency in inhibiting radical-induced red blood cell hemolysis was three times as high as the alpha-tocopherol efficiency. For beta-carotene no significant activity even in whole blood was shown. Highly surprising antioxidant activities were observed for acid folic and pyridoxine, compared to ascorbic acid. At 10 microM, the effectiveness of folic acid was almost three times as high as vitamin C. The biological test seems clinically more relevant than most other common assays because it can detect several classes of antioxidants.

Polymorphism of roxifiban.

Roxifiban was found to exist in two polymorphic forms. The polymorphs were detected by X-ray powder diffraction and solid-state carbon nuclear magnetic resonance. A slight difference between the two polymorphs was also detected by isothermal microcalorimetry. However, no differences were observed by differential scanning calorimetry, infrared, or Raman spectroscopy. Solubility studies as a function of temperature in a discriminating solvent system permitted characterization of the thermodynamics of the polymorphs. The enthalpy of solution at 25 degrees C was 8.1 kcal/mol and 8.9 kcal/mol for Form I and Form II, respectively, and the thermodynamic transition point was 132 degrees C. The data confirm that the polymorphs are enantiotropic. Form II is the thermodynamically stable crystal form over the practical range of drug substance storage and handling and dosage form processing and storage. However, Form I has been kinetically stable after storage for more than 36 months at 25 degrees C/60% relative humidity with no conversion to Form II occurring.

Solid-state carbon NMR characterization of the polymorphs of roxifiban.

Roxifiban, an experimental antithrombotic prodrug, exists as crystalline forms I and II. A quantitative solid-state nuclear magnetic resonance (NMR) method was developed to characterize the two polymorphs of roxifiban. The differences in the NMR spectra of the polymorphs were utilized in analyses of physical blends of the pure crystalline forms to establish a calibration curve. A detection limit of 9% form II in form I was determined from analysis of a 10% form II blend. Solid-state NMR was a valuable technique to quantify the polymorphic purity of roxifiban where other techniques such as differential scanning calorimetry (DSC) could not be used for this purpose.

Comparing the efficacy of two fluorescent retrograde tracers in labeling the motor and sensory neuron populations of the rat sciatic nerve.

We compared the efficacy with which the fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) retrogradely label neutrons. Trace crystals were applied to the sciatic nerve exclusively (single label) or serially (double label). Unbiased cell counts showed that FB and DY label similar numbers of motoneurons (P=1.00, df 5) or DRG neurons (P=0.95, df 5) when applied exclusively. Plotting of motoneurons revealed a similar pattern of distribution of FB and DY labeled neurons. When the tracers were applied serially, 79% of labeled motoneurons and 77% of labeled DRG neurons were double-labeled irrespective of which tracer was applied first. Equal proportions of the remaining labeled neurons were single-labeled with FB or DY. These data show that FB and DY label equal numbers of motor and sensory neurons of the sciatic nerve following exclusive or serial application of tracers. These findings support the use of FB and DY together in serial fluorescent labeling experiments.

Comparison of heterogeneous and homogeneous radioactivity flow detectors for simultaneous profiling and LC-MS/MS characterization of metabolites.

Methods for simultaneous liquid chromatography-radioactivity monitor (LC-RAM) metabolite profiling and LC-tandem mass spectrometry (MS/MS) characterization of metabolites are described. Profiling and characterization of metabolites from three drug candidates from different therapeutic areas were compared using in-line heterogeneous LC-RAM-MS/MS and homogeneous LC-RAM-MS/MS methods. Although comparison shows that simultaneous metabolite profiling and characterization can be achieved using either heterogeneous or homogeneous-LC-RAM-MS/MS systems, a homogeneous system has the advantage in the following aspects, (1) sensitivity; (2) ease of method transfer; (3) less peak broadening problems due to the drug or metabolites adhering to the RAM cell; (4) accuracy in quantitation of the metabolites; and (5) the ability to load larger volumes of unprocessed biological fluids. Furthermore, the study shows that some of the possible metabolites that do not ionize well with electrospray ionization (ESI) and eluded detection by heterogeneous-LC-RAM detection could be very easily detected and characterized using a homogeneous-LC-RAM-MS/MS system.

New applications of electron diffraction in the pharmaceutical industry: polymorph determination by using a combination of electron diffraction and synchrotron X-ray powder diffraction techniques.

Electron diffraction has been recently used in the pharmaceutical industry to study the polymorphism in crystalline drug substances. While conventional X-ray diffraction patterns could not be used to determine the cell parameters of two forms of the microcrystalline GP IIb/IIIa receptor antagonist roxifiban, a combination of electron single-crystal and synchrotron powder diffraction techniques were able to clearly distinguish the two polymorphs. The unit-cell parameters of the two polymorphs were ultimately determined using new software routines designed to take advantage of each technique's unique capabilities. The combined use of transmission electron microscopy (TEM) and synchrotron patterns appears to be a good general approach for characterizing complex (low-symmetry, large-unit-cell, micron-sized) polymorphic pharmaceutical compounds.

Detection and separation of the S-adenosylmethionine-decarboxylase inhibitor SAM486A in human plasma and urine by reversed-phase ion-pairing high-performance liquid chromatography.

A reversed-phase ion-pairing high-performance liquid chromatography method for the detection and separation of SAM486A in human plasma and urine is described. Precipitation of proteins was used for plasma sample preparation. Enrichment of SAM486A on a 5 micro C18 column using 0.05 M NaH(2)PO(4) and 0.005 M pentan-sulfonic acid (pH 3.0) as eluent was followed by isocratic elution onto a 5 micro C18 analytical column using 0.01 M NaH(2)PO(4) and 0.005 M pentan-sulfonic acid (pH 3.0) as eluent. Analysis time was 23.0 +/- 0.1 min. The separation parameters were: capacacity factor = 6.21; plates/m = 15,002; peak tailing = 2.076. The method is linear between 5 ng/ml (detection limit) and 1000 ng/ml.

Automated sample preparation of Roxifiban tablets: transfer of a manual method to an automated workstation.

Automation offers obvious advantages for the preparation of tablets prior to analysis by HPLC including unattended operation, minimization of human intervention and an electronic audit trail. However, significant effort has to be put in up front to develop and validate an automated method, particularly if it is required to closely follow an existing manual method. Here, method transfer for Roxifiban, a fibrinogen receptor antagonist, will be discussed. A Zymark tablet processing workstation II (TPWII) was used for all automated sample preparations. Manual methods for composite assay, content uniformity, weight variation and degradation products testing of a tablet formulation were transferred to the TPWII. The method involved weighing of the sample, disintegration of the dosage form by homogenization, extraction of the analyte in the homogenate solution, filtration of the homogenate, dilution of the filtrate and transfer to autosampler vials. Obstacles to a quick transfer included limitations in the volume capabilities of the TPWII, poor analyte solubility and achieving proper conditioning of the transfer lines and filter. After resolving these issues, a validated method was achieved. Spiked recoveries were from 99.4 to 101.1% (RSD's <0.5%). A cross-validation between automated and manual assay methods was compared by Westlake analysis giving a 0.7% calculated interval at the 95% confidence level. Carryover was 0.07% after 20 sample preparations at the highest tablet strength.