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Shoulder complex control of motion is influenced by neuromuscular function and can be quantified through the analysis of helical axes (HAs) dispersion. Muscle fatigue is a variable able to influence neuromuscular control, altering muscle activation timing and proprioception. The aim of the study was to describe shoulder complex HAs dispersion after muscle fatigue during upper limb movements of young healthy subjects. Thirty healthy right-handed volunteers (age 23.2 ± 2.6 years) were asked to perform a test made up of 15 humerothoracic flexion and rotation movements using both upper limbs in two different recording sessions. After each session, muscles of the tested movement were fatigued in isometric condition at dominant side. After fatigue, subjects repeated the test. Kinematics was recorded by an optoelectronic system and HAs dispersion was computed using Mean Distance (MD) and Mean Angle (MA) for the entire Range of Motion (RoM) and in portions of RoM. After fatigue of shoulder flexion muscles, greater MD (p = 0.001) and MA (p = 0.019) were found on the dominant side. After fatigue of shoulder rotation muscles, greater MD and MA were found on the dominant (p = 0.002 for MD; p = 0.047 for MA) and non-dominant (p = 0.038 for MD; p = 0.019 for MA) sides. Independently of fatigue, greater MA was found in portions of RoM with higher external resistance torque in flexion and rotation tasks. Muscle fatigue increases shoulder complex HAs dispersion, probably due to alteration in neuromuscular control. This data should be considered when exercise involving upper arms are proposed to subjects undergoing fatigue., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest related to the manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Knee joint rotation center displacement can be estimated in vivo through the analysis of helical axis (HAs) dispersion. HAs can be analyzed during walking, providing information on joint stability. The study aim was to describe knee HAs dispersion during walking in dominant and non-dominant legs of young and elderly healthy subjects. Twenty young (YG: age 23.3 ± 2.4 years) and twenty elderly (EG: age 69.3 ± 4.6 years) healthy subjects were asked to walk on a treadmill at a self-selected speed with reflective markers placed bilaterally on thighs and shanks to detect HAs dispersion and knee kinematics with an optoelectronic system. HAs dispersion was described during the following four phases of gait cycle: (1) flexion from 95% of the previous gait cycle to 10% of the subsequent gait cycle, (2) extension from 10% to 40%, (3) flexion from 40% to 75% and (4) extension from 75% to 95% of the gait cycle. Mean Distance (MD) and Mean Angle (MA) were used as HAs dispersion indexes during each gait phase. Participants showed greater MD and MA in sagittal and frontal planes during the first and second phases. EG revealed higher MD (p = 0.001) and MA (p < 0.001) during the first phase and higher MA (p = 0.001) during the fourth phase in both dominant and non-dominant legs on the sagittal plane. HAs dispersion could be related to the amount of forces acting on knee (first two phases) and knee degeneration (elderly). These results may be used as reference data in further studies on HAs dispersion in presence of knee pathologies or after knee surgery or rehabilitation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bee pollen is an attractive resource in the field of alternative remedies and thanks to the content of carbohydrates, crude fibers, proteins and lipids must be considered as a supplementary food of high potential rate. In characterization of bee pollen with the aim to define its value in human nutrition, the amino acids profile is one of the most important attributes. In the present study, the determination of amino acids composition of different monofloral bee pollen samples was obtained by an approach combining microwave acidic hydrolysis (60 min at 150 °C instead of 22 h at 120 °C in conventional oven) followed by derivatization using 9-fluorenylmethylchloroformate (FMOC-Cl) and separation of amino acids derivatives using a Phenomenex Kinetex core-shell 5 μm C18 (150 x 4.6 mm i.d.) column under a ternary gradient elution. Separation of 19 amino acids was achieved in about 40 min and fluorimetric detection (λ exc  = 265 nm λ em  = 315 nm) allowed selective and sensitive quantitation with LOQ values ranging within 0.14-3.00 μg/mL. Interestingly, the present approach allowed determination of some amino acids e.g., tryptophan and trans-4-hydroxyproline that are often lost by other methods of analysis. Significant differences in the composition of the considered samples were found confirming the impact of botanical origin of the product on its nutritional value. Principal Component Analysis was applied to treat the obtained data, highlighting the importance for discrimination, of detecting low abundance amino acids. The proposed method can be used as an advantageous alternative to the existing ones for characterization of bee pollen as an important source of dietary proteins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The shoulder complex (SC) consists of joints with little congruence and its active and passive structures ensure its stability. Stability of the SC rotation centre during upper arm movements can be estimated through the analysis of Helical Axes (HAs) dispersion. The aim of this study was to describe shoulder HAs dispersion during upper limb movements performed with dominant and non-dominant arms by young and elderly subjects. Forty subjects participated in the study (20 young: age 24.8 ± 2.8 years and 20 elderly: age 71.7 ± 6.3 years). Subjects were asked to perform four cycles of 15 rotations, flexions, elevations and abductions with one arm at a time at constant speed. Reflective markers were placed on participants' arms and trunk in order to detect movements and the HAs dispersion with an optoelectronic system. Mean Distance (MD) from the HAs barycenter and Mean Angle (MA) were used as HAs dispersion indexes. Young subjects showed significant lower MD compared to the elderly during all motion ranges of rotation, flexion and elevation (p < 0.001). Moreover, the MD was lower in the dominant arm compared to the contralateral for rotation (p = 0.049) and flexion (p = 0.019). The results may be due to joint degeneration described in elderly subjects and differences in neuromuscular control of SC stability., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Amino acids playing important roles in metabolic processes are often included in dietary supplements whose use has largely expanded over the last 20 years not only in patients with particular deficiencies, but also in athletes and even common people that want to enrich their regular daily diet. In the present study, a bare silica Kinetex core-shell 2.6μm HILIC column was used for separation of some important hydrophilic amino acids and amino acids-like molecules i.e., aspartic acid, creatine, carnitine, arginine and the tripeptide glutathione (GSH), by optimizing the chromatographic conditions for their determination in complex alimentary supplements. The contribution of partition, adsorption and ion exchange on the retention mechanism was studied by varying parameters such as water content and the counter-ion concentration in the mobile phase. Optimum conditions employed a Phenomenex Kinetex core-shell 2.6μm HILIC (100×4.6mm i.d.) column and a mobile phase of acetonitrile/potassium phosphate buffer (12.5mM; pH=2.8) 85:15, v/v, at the flow rate of 1.4mL/min, using UV detection at 200nm. A reference HPLC method for the selective determination of GSH by using 1,4-naphthoquinone as derivatization reagent was also introduced for comparative purposes. The developed HILIC method was validated and applied to the analysis of the considered compounds in dietary supplements. Interestingly, in some of the real samples, oxidized glutathione which is an inactive impurity of GSH, was found at the level of about 20%. The proposed study confirms the importance of simple analytical methods for a rigorous quality control of dietary supplements containing unstable active ingredients., (Copyright © 2017 Elsevier B.V. All rights reserved.)

1,4-Anthraquinone (ANQ) is proposed as a novel pre-column reagent for high performance liquid chromatography (HPLC) determination of N-acetylcysteine (NAC) and captopril (CAP) in pharmaceutical formulations. The derivatization reactions were carried out at room temperature: NAC at pH 8 for 1min, while CAP at pH 7.5 for 20min. Both reactions reached completeness at a reagent to thiol molar ratio of about 2.5. The synthesised derivatives were characterized by 1 H NMR and IR. The chromatographic separations were performed on a C 18 Phenomenex Synergi Fusion 4μm (250mm×4.6mm I.D.) stainless steel column with detection at λ=300nm. The mobile phase consisted of methanol/triethylammonium (TEA) phosphate buffer (pH 3; 0.05mol/L) 75:25 (v/v) at a flow-rate of 0.4mL/min for NAC and 88:12 (v/v), at a flow-rate of 0.6mL/min for CAP. The validation parameters (linearity, sensitivity, accuracy, precision, specificity and stability) were highly satisfactory. Linear response was observed (determination coefficient ≥0.9996). Detection limits were about 8 and 18ng/mL for NAC and CAP, respectively. Intra-day precision (relative standard deviation, R.S.D.) was ≤1.58%, for thiol to internal standard (IS) peak area ratio and ≤0.33%, for thiol and IS retention times (t R ), without significant differences between intra- and inter-day data. Thiol recovery studies were satisfactory (99.50%) with R.S.D. ≤0.56%. The results highlight the high sensitivity of the method and the remarkable reactivity and selectivity of the reagent towards the thiol function. The developed method is suitable for the quality control of both thiols in commercial products. The method can be applied in any analytical laboratory not requiring a sophisticated instrumentation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A simple and fast chromatographic method using ultraviolet diode-array detector (UV-DAD) was developed for the automatic high performance liquid chromatography (HPLC) determination of the title of oleuropein in a new dietary supplements in form of effervescent granules. The chromatographic separations were performed on a C18 core-shell column with detection at λ=232nm. The mobile phase consisted of deionized water with 0.1% TFA and acetonitrile under gradient conditions at a flow-rate of 0.8mL/min. Oleuropein and oleuroside present in the raw material were characterized by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The validation of the analytical procedure has been performed determining the following parameters: specificity, linearity, repeatability, reproducibility, accuracy, limit of quantification (LOQ), stability of the standard and sample solutions. Linear response was observed in fortified placebo solutions (determination coefficient: 0.9998). Intra-day precision (relative standard deviation, RSD) was ≤5.0% for peak area and for retention times (tR) without significant differences between intra- and inter-day data. The limits of quantitation (LOQ) was about 5μg/mL and 9pmol/inject. Oleuropein recovery studies gave good results (99.9%) with a R.S.D. of 0.5%. The speed of analysis and the stability of the solutions with a fluctuation Δ (%) ≤2.0 at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of many samples and consecutive chromatographic analyses by using an autosampler. The developed method is suitable for the quality control of oleuropein in raw material and industrial products. The method can be applied in any analytical laboratory not requiring a sophisticated instrumentation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Iodinated radiocontrast media (RCM) are usually well tolerated, but their large and increasing use renders their toxicity a relevant problem, especially in high risk patients. The aim of the study was to investigate the possible toxic or activating effects of iodixanol on endothelial cells (EC) and the putative in vitro protective action of N-acetylcysteine and rosuvastatin. Morphology, oxidative status, redistribution of heat-shock protein 60 and secretion of proinflammatory products were studied in cultured human EC through confocal microscopy, immunofluorescence and immuno-enzymatic methods. EC reacted to iodixanol with shrinking and bulging, increase in intracellular oxidation and translocation of Heat Shock Protein 60 to the cell membrane. The secretion of proinflammatory products was strongly stimulated by sequential incubation of EC with iodixanol and TNFα (p<0.00001 for all tested molecules, namely TNFα, IL-8, sVCAM-1, MCP-1, and IL-6). N-acetylcysteine prevented morphologic and oxidative derangements, and significantly reduced proinflammatory product secretion (P range<0.0001 to<0.00001 for TNFα, VCAM-1, MCP-1, and IL-6); rosuvastatin inhibited morphology and oxidative modifications only. Our data help clarifying the mechanisms of early and late toxicity of RCM and support the use of anti-oxidant and anti-inflammatory agents for optimization of radiological procedures in high risk patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In this report we focused our interest on the early events of the replication cycle of NWS/33 human influenza A (NWS) virus in MDCK (canine), LLC-MK2 (simian), and NSK (swine) kidney cells, with different susceptibility upon infection. We have previously demonstrated that actin organization induces restriction to viral replication during the early stages of NWS virus infection in simian kidney cells. To explore how cell endocytic mechanisms are hijacked by NWS virus and may modulate the outcome of viral infection, the effect of drugs affecting selectively the entry via clathrin-coated pits, caveolar/raft-dependent endocytosis and macropinocytosis was analyzed. Results point to critical differences in terms of internalization pathways exploited by NWS virus to enter the examined cell models. Moreover, we show that some ways of entry do not allow an effective virus internalization, depending on the cell type. Understanding how specific cell functions/components may regulate early phases of viral replication allows us to deepen our knowledge on influenza virus infection and provides new insights for anti-viral researches., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The use of 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde as a precolumn derivatization reagent for HPLC analysis of amino acids is proposed. The compound reacts under mild conditions (10min at ambient temperature) with primary amino groups. The derivatization conditions to obtain quantitative reaction were optimised by considering different parameters (temperature, pH and reagent concentration) using l-Val as the model compound. The synthesized l-Val derivative was characterized by (1)H NMR and UV. The derivatives of 19 amino acids were separated by reversed-phase HPLC and detected at lambda=320nm. The method was applied successfully to the qualitative and quantitative analysis of commercial polyamino acid preparations.

Excitatory amino acid carrier 1 (EAAC1) belongs to the family of the Na(+)-dependent glutamate carriers. Although the association between defective EAAC1 function and neurologic disease has been repeatedly studied, EAAC1 regulation is not yet fully understood. We have reported that in C6 glioma cells both the activity and membrane targeting of EAAC1 require the integrity of actin cytoskeleton. Here we show that, in the same model, EAAC1 partially co-localizes with actin filaments at the level of cell processes. Moreover, perinuclear spots in which EAAC1 co-localizes with the actin binding protein alpha-adducin are observed in some cells and, consistently, faint co-immunoprecipitation bands between EAAC1 and alpha-adducin are detected. Co-localization and partial co-immunoprecipitation of EAAC1 and adducin are still detectable after cell treatment with phorbol esters, a condition that leads to a protein kinase C (PKC)-dependent increase of EAAC1 expression on the membrane and to the phosphorylation of adducin. A co-immunoprecipitation band was also detected in protein extracts of rat hippocampus. The amount of adducin co-immunoprecipitated with EAAC1 increases after the treatment of C6 cells with retinoic acid, a differentiating agent that induces EAAC1 overexpression in this cell model. Moreover, in clones of C6 cells transfected with a hemagglutinin (HA)-tagged adducin, the bands of EAAC1 immunoprecipitated by an anti-HA antiserum were proportional to EAAC1 expression. These results suggest the existence of a pool of EAAC1 transporters associated with the actin binding protein alpha-adducin in a PKC-insensitive manner., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

The overview of cortisol physiology, action and pathology is achieved in relation to the hypothalamic-pituitary-adrenal axis alteration by laboratory investigation. The measurements of cortisol and related compound levels in blood, urine and saliva used to study the physiological and pathological cortisol involvement, are critically reviewed. The immunoassay and chromatographic methods for cortisol measurement in the various biological fluids are examined in relation to their analytical performances, reference ranges and diagnostic specificity and sensitivity. Moreover, blood, urine and saliva cortisol level measurements are described taking into account the diagnostic implications. The deduction is that each method requires the definition of its own reference range and its related diagnostic cut-off levels. Thus, this review, stressing the analysis procedures, could help to understand and compare the results of the different assays.

Furoin, a benzoin analogue, was examined as novel fluorogenic reagent for the selective and sensitive LC determination of various guanidines after pre-column derivatization. The derivatization reaction was carried out at 100 degrees C for 5 min to give adducts that were separated on a Phenomenex Synergi MAX-RP column and detected at lambda(em)=410 nm with lambda(ex)=325 nm. The reagent showed to be useful both for determining together arginine (Arg) and creatine (CT) in dietary supplements under elution isocratic conditions and for the simultaneous analysis of a variety of guanidines in biological samples (human plasma and urine) under elution gradient conditions. The detection limits ranged from 7 to 25 fmol. Recovery studies showed good results for all determined guanidino compounds (85.6-106.2%; R.S.D.=1.1-6.2%).

A reversed-phase ion pair liquid chromatographic method (RP-LC) for the determination of dehydroascorbic acid (DHA) and ascorbic acid (AA) and also acetaminophen, which is combined in pharmaceuticals, is proposed and validated. AA and acetaminophen were analyzed directly, while DHA was determined after pre-column derivatization with 4,5-dimethyl-1,2-phenylenediamine (DMPD). The derivatization reaction was carried out under mild conditions (10min at ambient temperature) in the dark in sodium acetate buffer (80mM; pH 3.7) solution containing EDTA as metal scavenger. The chromatographic separations were performed on a Phenomenex Synergi 4u hydro-RP (150mmx4.6mm) under isocratic elution conditions, using cetyltrimethylammonium bromide (CTAB) as ion-pairing reagent in the mobile phase. Linear responses were observed for each compound. The intra-day precision (R.S.D.) was < or =1.40% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results for all compounds (99.7-101.8%) with R.S.D. ranging from 0.56 to 1.82%. The limits of quantitation were about 40, 50 and 140pmol for acetaminophen, AA and DHA, respectively. The DHA impurity values found in dosage forms were < or =0.2% of AA.

A reversed-phase liquid chromatographic method (RP-LC) is proposed and validated for the analysis of branched-chain amino acids (l-leucine, l-isoleucine and l-valine) in new pharmaceutical formulations. The pre-column derivatization reaction of these amino acids with 2,4-dinitrofluorobenzene (DNFB) has been investigated considering the matrix effect. The compound reacts at 60 degrees C for 10 min at pH 9 with the amino function, in presence of cetyltrimethylammonium bromide (CTAB), to give adducts that have been separated on a RP amide C16 column and detected at lambda=360 nm. Linear responses were observed for each derivative. The intra-day precision (R.S.D.) was

Objectives: Adrenergic alpha(1)-receptors agonists and antagonists have been reported to increase and reduce, respectively, neurogenic inflammatory responses mediated by capsaicin-sensitive sensory neurons. However, the precise role and localization of the alpha(1)-adrenoceptors involved in these effects are not known., Methods: We have studied in the rat whether functional alpha(1)-adrenoreceptors are expressed in primary sensory neurons, and whether they regulate neurogenic inflammation and nociceptive responses in the urinary bladder., Results: The alpha(1)-adrenoreceptor agonist phenylephrine (1 micromol/l) (1) mobilized intracellular Ca(2+) in cultured lumbar and sacral dorsal root ganglia neurons, (2) caused the release of substance P (SP) from terminals of capsaicin-sensitive sensory neurons from the lumbar enlargement of the dorsal spinal cord and urinary bladder, and (3) increased plasma protein extravasation in the urinary bladder. All these effects were abolished by the alpha(1)-adrenoceptor antagonist alfuzosin (10 micromol/l). Furthermore, alfuzosin (30 microg/kg, i.v.) partially, but significantly, inhibited cyclophosphamide-induced plasma protein extravasation in the rat urinary bladder. Phenylephrine-induced Ca(2+) mobilization in cultured dorsal root ganglia neurons was exaggerated by pretreating the rats in vivo with cyclophosphamide. Finally, cyclophosphamide increased c-fos expression in the rat lumbar spinal cord. Also these in vitro and in vivo effects were inhibited by pretreatment with alfuzosin., Conclusions: Alpha(1)-adrenoceptors are functionally expressed by capsaicin-sensitive, nociceptive, primary sensory neurons of the rat urinary tract, and their activation may contribute to signal irritative and nociceptive responses arising from the urinary tract. It is possible that, at least, part of the beneficial effects of alpha(1)-adrenoceptor antagonists in the amelioration of storage symptoms in the lower urinary tract derives from their inhibitory effect on neurogenic inflammatory responses.

Objectives: To examine an immunoassay for measuring free IGF-I in a saliva specimen (free sIGF-I) and to study the levels in relation to the training conditions comparing young athletes and sedentary females., Design and Methods: The analysis was carried out by modifying a commercial kit for plasma matrix to measure the free sIGF-I. The plasma free and total IGF-I fractions, hGH and salivary total proteins were also measured. Saliva and blood specimens were collected from 15 well-trained young female volleyball athletes and from a control group of 14 young sedentary females., Results: The calibration curve to assay free sIGF-I covered the range 0.05-5.00 microg/L. The detection limit was 0.07 microg/L. The within-run and between-run imprecision CVs were 10% and 13% respectively. The average recovery was 88%. Free sIGF-I, measured in 15 athletes and in 14 young sedentary females, was 0.10+/-0.03 and 0.20+/-0.05 micarog/L respectively (p<0.001)., Conclusions: There were decreased levels of free sIGF-I in well-trained athletes, compared with sedentary females. This decrease could be related to a greater tissue requirement by the active muscle subjected to intense exercise for several days.

The use of anisoin as pre-chromatographic reagent for LC analysis of guanidino compounds is proposed. The reagent reacts (5 min at 100 degrees C) with guanidino function and the resulting adducts can be chromatographed under reversed-phase conditions. A fluorescence detector (lambda(ex)=325 nm; lambda(em)=435 nm) was used to detect guanidino adducts. The derivatization and chromatographic conditions were optimised by a series of experiments. Application to the determination of arginine and creatine in pharmaceuticals and arginine, guanidine, methylguanidine, guanidinosuccinic acid, beta-guanidinopropionic acid, gamma-guanidinobutyric acid, guanidinoacetic acid and homoarginine in human urine is described. Quantitation limits ranged from 6 to 30 fmol, except for creatine (510 fmol).

A sensitive LC method for the determination of leuprolide acetate component amino acids in injectable solution with fluorogenic pre-column derivatization has been developed. The derivatization reaction with phanquinone was optimised by a series of experiments. Histidine, arginine, serine, tryptophan, glutamic acid, tyrosine, methionine, isoleucine, leucine and phenylalanine were separated on a reversed-phase ODS column using as eluent a binary mixture of triethylammonium phosphate buffer-methanol, under gradient elution conditions. The derivatives were eluted in 30 min with good reproducibility. The hydrolysis reaction of the peptide was carried out at reflux with 12 N hydrochloric acid for 2 h 30 min. The intra-day accuracy of the entire procedure (hydrolysis, derivatization, LC separation) ranged from 80.5 to 109.5% of the nominal concentration of leuprolide acetate and the precision (%R.S.D.) was less than 5.8%; the inter-day accuracy was in the range 81.5-107.2% and corresponding R.S.D. values were less than 4.6%. The detection limits (signal-to-noise ratio=3) for the adducts are 30-800 fmol.

4,7-Phenanthroline-5,6-dione (phanquinone) was used as a fluorogenic labeling reagent in pre-column derivatization for the quality control of amino acids in pharmaceuticals. The amino acid adducts were efficiently separated by C12 RP high-performance liquid chromatography (HPLC) using a ternary mixture of triethylamine (TEA) phosphate buffer (pH 2.5, 0.05 M)-methanol- tetrahydrofuran (THF) as mobile phase by varying composition gradient elution and detected fluorometrically. The results obtained by the proposed method were compared statistically, by means of the Student's t-test and the variance ratio F-test, with those obtained by a rapid reference method, which involved o-phthaldialdehyde (OPA) as pre-column reagent; no significant difference was found. The stronger derivatization conditions (60 degrees C, pH 8, 60 min) required for the method with phanquinone are compensated by the major stability of derivatives and by the absence of fluorescent degradation products.

A spectrophotometric method (lambda=535 nm) for the iron (III) impurities determination in iron protein-succinylate complex syrup using thioglycolic acid in basic ambient was proposed and validated. Assay samples were treated with 0.1 N hydrochloric acid and centrifuged to remove the interfering active drug. Linear response (r=0.9999) was observed over the range of 0.005-0.2% of the iron (III) with respect to the complex nominal concentration. The accuracy could be considered very satisfactory (recovery=97-99%). The intra-day precision (RSD) of impurity amongst six independent sample preparations, was 1.4%, and there was no significant difference between intra- and inter-day studies. Intermediate precision indicated that the assay possessed high degrees of ruggedness. The limit of quantitation was 0.005% of impurity with respect to the active drug. The results obtained for iron (III) were compared statistically with those obtained with the standard addition method by means of the Student's t-test and the variance ratio F-test; no significative difference was found.

Liquid chromatographic (HPLC) methods with fluorescence detection at different wavelengths were developed for measurements of retinoic acids (13-cis and all-trans) in pharmaceutical dosage forms and components of 'retinoid solution' (all-trans retinoic acid, vitamin A palmitate and beta-carotene), a galenical of 'Di Bella therapy', using reversed phase columns under isocratic conditions. The stability of all-trans retinoic acid in cream and all-trans retinoic acid and vitamin A palmitate in 'retinoid solution' was investigated. Solid-phase extraction (SPE), using C18 sorbent was applied to the analysis of retinoic acids (9-cis, 13-cis and all-trans) in the 'retinoid solution' to obtain a practical and reliable sample clean-up. The results showed that these preparations (cream and solution) can be conveniently stored in the dark (t.a. or 2-8 degrees C): under these conditions about 86-87% of the all-trans retinoic acid initial concentration in both formulations and about 73-78% of vitamin A palmitate in the 'retinoid solution' remained after 90 days, while under sunlight exposure rapid degradation of the drugs was observed.

To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.

Objective: For 4 years we have monitored the histological evolution of ileal neobladders in a single cohort of 30 patients in order to systematically describe the histological changes occurring after surgery. The aim of the study was to evaluate the long-term evolution of many histological parameters with functional relevance as to the metabolic outcome of the reservoirs., Methods: Ileal samples were collected during surgery and by random biopsies during cystoscopy 6, 12, 18, 24, 36 and 48 months later. At each step qualitative and quantitative assessment of the histological and cytological conditions of the samples was carried out., Results: Morphological changes develop relatively early but the situation tends to level out in about 1 year. The morphological changes are topographically uneven and, although mucosal flattening becomes progressively prevalent, areas with shortened villi persist indefinitely. Goblet cells prevail over enterocytes and the secretive pattern shifts towards sialomucins. The overall replication rate decreases initially but tends to restore in 1 year. Dysplasia or atrophy were never recorded., Conclusions: The 4-year systematic follow-up revealed a typical histological adaptation pattern in the ileal neobladder without signs of dysplasia. The changes seem to be induced by the aggressive environment and develop in the time lag required for functional adaptation of the epithelium., (Copyright 1999 S. Karger AG, Basel.)

A supercritical fluid extraction (SFE) method was developed for the isolation of imidazole antimycotic drugs (miconazole, econazole, clotrimazole and bifonazole) from cream preparations. The SFE process involved static (1 min) and dynamic (4 min) extraction steps using pure and 10% methanol modified carbon dioxide. The SFE step was then followed by derivative UV spectrophotometric analysis. The method proved to be suitable for quality control assays of the examined antimycotics in commercial cream formulations.

A fluorimetric liquid chromatographic method (lambda(ex) = 280 nm; lambda(em) = 312 nm) was developed for measurements of unconjugated estrogens (estradiol and estriol) in pharmaceutical dosage forms using a reversed-phase column with water acetonitrile at different composition as mobile phase. The in vitro release profiles of three different estradiol transdermal therapeutic systems were determined through a medical-grade silicone rubber subdermal implant material membrane, using a modified Franz diffusion apparatus at 37 degrees C in presence of PEG 400. The HPLC method possesses advantages of rapidity, simplicity and accuracy.

The use of HPLC with fluorescence detection (lambda ex = 280 nm; lambda em = 410 or 312 nm) in combination with a postcolumn on line photochemical derivatization was investigated for the analysis of conjugated and unconjugated estrogens and their correlated impurities. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo induced modifications were useful for the identification of the various estrogens. The proposed HPLC methods were successfully applied to the analysis of commercially available conjugated estrogens (raw materials and pharmaceuticals) and estrogen samples. The assays results relative to the pharmaceutical formulations were in agreement with those obtained by a reference HPLC method with UV detection (lambda = 280 nm).

The use of 2-chloro-6,7-dimethoxy-3-quinolinecarboxaldehyde as a fluorogenic labelling reagent in pre-column derivatization for the HPLC separation of chlorophenols has been investigated. The compound reacts (50 min at 110 degrees C) with 2- and 4-chlorophenols to give fluorescent ethers that can be separated by reversed-phase HPLC and detected at lambda exc = 360 nm, lambda em = 500 nm. The experimental conditions for derivatization and chromatographic separation are discussed. Applications for the determination of chlorocresol (4-chloro-3-cresol) and chloroxylenol (4-chloro-3,5-xylenol) in pharmaceutical formulations (creams, ointments) are described.

Solid-phase extraction (SPE) using C-18, diol and ion-exchange sorbents followed by UV spectrophotometric (conventional and derivative mode) assay was applied to the analysis of basic, acidic and neutral drugs commercially available in creams. A representative set of drugs (promethazine, chlorhexidine, benzydamine, ketoprofen, ibuprofen, fentiazac, piroxicam, fluorouracil, crotamiton and hydrocortisone acetate) was selected, and for each drug the appropriate SPE conditions (adsorption, washing and elution) were investigated to obtain a practical and reliable sample clean-up. It was shown that the developed SPE procedures were capable of removing interfering cream components (excipients including preservatives) allowing accurate spectrophotometric analyses to be performed. In some applications, derivative spectrophotometry was advantageous over the conventional absorption mode with respect to higher selectivity and versatility.

Unlike conventional potentiometric goniometers, flexible electrogoniometers do not suffer from any alignment problems with respect to the joint axis. We hypothesized that flexible goniometers provide more valid measurements in that they avoid some biomechanical pitfalls. With both devices simultaneously we measured the movements of dorsal flexion of the ankle and flexion of the knee in three healthy subjects. In the various subjects, this comparison showed that the flexible goniometers signalled ankle excursion greater by 19-40% with the foot remaining in neutral position or being simultaneously pronated, and lower by 10-21% with the foot being simultaneously supinated. At the knee the flexible goniometers signalled a flexion greater by 24-32%, with respect to potentiometric goniometers. Biomechanical considerations support the validity of records taken with flexible goniometers, potentiometric measures being biased by (a) multijoint, multiplane motions underlying foot dorsal flexion, and (b) multiaxis motion underlying knee flexion RELEVANCE: In measurements of angles between adjacent body segments, flexible goniometers should be preferred to potentiometric goniometers despite their higher cost. Flexible goniometers not only are more practical: more importantly, they provide a valid measure of relative orientations in one plane, regardless of the number and different concurrent motions of the underlying joints.

2-Bromoacetyl-6-methoxynaphthalene has been found to be a useful prechromatographic fluorescent labelling reagent for the analysis of dicarboxylic acids. The derivatization reaction of azelaic acid and meglutol with this reagent yieded stable and highly fluorescent diesters which could be analysed by reversed-phase HPLC with fluorescence detection. According to the nature of the sample, the derivatization reaction could be carried out in acetonitrile or in an aqueous micellar system. The proposed methods proved to be suitable for the quality control of various complex pharmaceutical and cosmetic formulations of the azelaic acid and meglutol.

The use of HPLC with different detectors has been investigated for the analysis of bile acid impurities present in four different commercially available ursodeoxycholic acid preparations. The bile acids were efficiently separated by C18 reversed-phase HPLC using methanol-water (3:2, v/v) as the mobile phase. The detectors used for bile acid detection were: UV at 200 nm refractive index (RI) and an evaporative light scattering mass detector (ELSD II). A prederivatization method with the formation of a fluorescent naphthacyl ester has also been used. GC-MS analysis of Me-TMS bile acid derivatives was included as a reference method. The four ursodeoxycholic acid samples were 98-99% pure. The main impurities present in the samples were chenodeoxycholic acid and to a lesser extent lithocholic acid. Only one sample was found to be almost 100% pure using all the detectors. Significant agreement of the data was found between RI, ELSD II detectors and the fluorescent method; the UV detector was unsuitable for use in this method. The analytical performances of the four detectors for bile acid analysis are reported and discussed. When the four-detector data were compared with the GC-MS method, reasonable agreement resulted. Discordant results were found in the quantitation of trace impurities like lithocholic acid and/or other minor bile acids present in amounts less than 0.1%.

2-Bromoacetyl-6-methoxynaphthalene was used as a pre-chromatographic fluorescent labelling reagent for the high-performance liquid chromatographic (HPLC) analysis of bile acids. The derivatization reaction was performed in an aqueous medium in the presence of tetrahexylammonium bromide by ultrasonication at 40 degrees C to give fluorescent esters which were separated by reversed-phase HPLC and detected fluorimetrically (lambda ex = 300 nm, lambda em = 460 nm). Applications to the determination of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) in their pharmaceutical formulations are described. The method was also applied to the determination of free and conjugated bile acids in human bile samples.

Reversed-phase HPLC on different column packing materials (Hypersil C-18, Spherisorb-CN, Chromspher-B) is used to obtain selective separations of imidazole antimycotic drugs, such as ketoconazole, clotrimazole, tioconazole, bifonazole, isoconazole, econazole, miconazole and fenticonazole. The use of a post-column on-line photochemical reactor is shown to be useful for the enhancement of the sensitivity of the HPLC analysis with UV detection. The proposed HPLC methods are applied to the analysis of commercial dosage forms (creams) with solid-phase extraction (SPE) procedure, using a diol sorbent, being found to be a convenient technique for the sample preparation giving quantitative drug recovery.

Methods based on derivative UV spectrophotometry and high-performance liquid chromatography (HPLC) have been developed for the selective determination of miconazole and econazole in pharmaceutical dosage forms. A solid-phase extraction (SPE) procedure using a diol column gave quantitative drug extraction from formulated creams and provided purified sample solutions suitable for assay by the derivative UV spectrophotometric and HPLC methods. The proposed methods gave comparable accurate results, whereas a conventional UV spectrophotometric method was found to be seriously affected by excipients.