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Multi-linear gradients are a convenient solution to get separation of complex samples by modulating carefully the gradient slope, in order to accomplish the local selectivity needs for each particular solute cluster. These gradients can be designed by trial-and-error according to the chromatographer experience, but this strategy becomes quickly inappropriate for complex separations. More evolved solutions imply the sequential construction of multi-segmented gradients. However, this strategy discards part of the search space in each step of the construction and, again, cannot deal properly with very complex samples. When the complexity is too large, the only valid alternative for finding the best gradient is the use of global search methods, such as genetic algorithms (GAs). Recently, a new global approach where the level of detail is increased along the search has been proposed, namely Multi-scale optimisation (MSO). In this strategy, cubic splines are applied to build intermediate curves to define any arbitrary solvent variation function. Subdivision schemes are used to generate the cubic splines and control their level of detail. The search was subjected to a number of restrictions, such as avoiding long elution and favouring a balanced peak distribution. The aim of this work is evaluating and comparing the results of GAs and MSO. Both approaches were tested with a set of 14 diuretics and probenecid, eluted with acetonitrile-water mixtures using a C18 column. Satisfactory baseline resolution was obtained with an analysis time of 15-16 min. We found that GAs optimisation offered results equivalent to those provided by MSO, when the penalisation parameters were included in the cost function., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Highly selective molecularly imprinted polymers (MIPs) for solid-phase extraction and determination of furosemide in human plasma have been designed and prepared. In order to study the intermolecular interactions in the pre-polymerization mixture and to find a suitable functional monomer in MIP preparation, a computational approach was developed. It was based on the comparison of the binding energy of the complexes between the template and functional monomers. Having confirmed the results of computational method, three MIPs were synthesized with different functional monomers, i.e. acrylamide (AAM), 4-vinylpiridine (4-VP) and acrylonitrile (ACN), and then evaluated using Langmuir-Freundlich (LF) isotherm. Using the MIP prepared by AAM as functional monomer, a molecularly imprinted solid-phase extraction procedure followed by high performance liquid chromatography with ultraviolet detector (MISPE-HPLC-UV) was developed for selective extraction and determination of furosemide in human plasma. For the proposed MISPE-HPLC-UV method, the linearity between responses (peak area) and concentration was found over the range of 75-3500 ng mL(-1) with a linear regression coefficient (R(2)) of 0.997. The limit of detection (LOD) and quantification (LOQ) in plasma were 12.9 and 43.3 ng mL(-1), respectively., (Copyright 2009 Elsevier B.V. All rights reserved.)

Peak capacity (i.e. maximal number of resolved peaks that fit in a chromatographic window) is a theoretical concept with growing interest, but based on a situation rarely met in practice. Real chromatograms tend to have uneven distributions, with overlapped peaks and large gaps. The number of resolved compounds should, therefore, be known from estimations. Several equations have been reported for this purpose based on three perspectives, namely, the intuitive approach (peak capacity as the size of the retention time window measured in peak width units), which assumes peaks with the same width, and the outlines of Giddings and Grushka, which consider changes in peak width with retention time. In this work, the peak capacity concept is discussed and three new approaches are derived based on realistic descriptions of peak shape. The first one is based on the Grushka's approach and considers the contributions of column and extra-column peak variances. The second one relies on Giddings' and assumes asymmetrical peaks where left and right peak half-widths depend linearly on retention time. The third equation, based on the intuitive approach, uses a mean peak width obtained by integration, instead of a mean value from several representative peaks. The accuracy of the classical Giddings' approach for ideal peaks, a modification of the Grushka's approach that considers variation of peak width at half-height, and the three new approaches were checked on combined chromatograms built by adding real peaks. The results demonstrate that the change in efficiency (and not in skewness) is the relevant factor, at least in the studied examples. Also, peak width should be measured at low peak height ratios (i.e. 10%) to better account peak deformation.

The organic solvent content and the pH in the mobile phase are the usual main factors in reversed-phase liquid chromatographic separations, owing to their strong effects on retention and/or selectivity. Temperature is often neglected. However, even in cases where the impact of this factor on selectivity is minor, the reduction in analysis time is still an interesting reason to consider it. In addition, ionisable compounds may exhibit selectivity changes, owing to the interaction of organic solvent and/or temperature with pH. The separation of ionisable compounds (nine diuretics: bendroflumethiazide, benzthiazide, bumetanide, chlorthalidone, furosemide, piretanide, probenecid, trichloromethiazide and xipamide, and two beta-blockers: oxprenolol and propranolol) exhibiting different acid-base behaviour was studied. The compounds were tested in a Zorbax SB C18 column under a wide range of conditions: 25-45% (v/v) acetonitrile, pH 3-7 and 20-50 degrees C. Models considering two factors (organic solvent/pH and temperature/pH), and three factors (organic solvent/temperature/pH) were developed from a previously reported equation, which considers the polarity contributions of solute, stationary and mobile phases. This allowed a comprehensive method to predict the retention of the 11 compounds, the modification of their acid-base behaviour (i.e. determination of protonation constants and shifts of the retention versus pH curves), and the selectivity changes within the studied factor ranges.

Torasemide is a "loop type" diuretic drug. For pharmacokinetic studies sensitive analytic methods are essential for authentic qualitative and quantitative information. A robust, selective and sensitive HPLC method is described for the simultaneous determination of torasemide, its major metabolite M5 and its active metabolites M1 and M3 and an internal standard within 17 min. Solid-phase extraction with C(2)-cartridges was used for the clean-up of plasma samples. The chromatographic separation was carried out on a CN-column with a mobile phase consisting of perchloric acid (0.02 M, pH 2.5)/acetonitrile (90/10, v/v)). The calibration range used reached from 20 to 1000 ng/ml for all analytes. Coefficients of variation were less than 10% at every calibration point for each analyte. Plasma concentrations in samples obtained from volunteers in the course of a clinical study could be reliably measured with this method. Median maximum concentrations in plasma after a 10mg oral dose during a 24h study interval were located at 1h for torasemide, 1h for M1 and 2h for M5. Concentrations between 2226 and <20 ng/ml for torasemide, between 159 and <20 ng/ml for M1 and between 420 and <20 ng/ml for M5 were observed.

Ethanolic extract of Rungia repens aerials parts (300 and 600 mg/kg p.o) showed diuretic activity in rats. The acute toxicity, orally evaluated in mice, was found to be higher than 3000 mg/kg.

A comparative study on the performance of two RPLC modes on the separation of 18 diuretics with diverse acid-base behaviour (acetazolamide, althiazide, amiloride, bendroflumethiazide, benzthiazide, bumetanide, canrenoic acid, chlorothiazide, chlorthalidone, ethacrynic acid, furosemide, hydrochlorothiazide, piretanide, probenecid, spironolactone, triamterene, trichloromethiazide and xipamide) was carried out. A conventional octadecylsilane column and acidic acetonitrile-water mobile phases, in the absence and presence of micelles of the anionic surfactant sodium dodecyl sulphate (SDS), were used. The effects of pH and the modifiers acetonitrile and SDS on peak asymmetry, efficiency, selectivity, resolution and analysis time, were examined. The comparison of both RPLC modes (aqueous- and micellar-organics) was done using the same processing tools, applying several polynomial and mechanistic equations to describe the retention. The best separations were obtained by maximising the product of peak purities, considering a wide range of experimental conditions. The study illustrates that, despite the theoretical and practical complexity of the problem, the predicted optimal chromatograms can be reproduced experimentally with great accuracy. None of the examined RPLC modes was able to yield baseline separation of the 18 diuretics. However, their selectivity was complementary, being appropriate for different combinations of a smaller number of the assayed diuretics.

In this work, a rapid cyclodextrin-assisted capillary electrophoretic method is developed for the separation of the diuretic torasemide and three of its metabolites. Both fractional factorial and central composite designs were employed to optimise the separation method. The factors studied were pH, concentration of methyl-beta-cyclodextrin, concentration of the background electrolyte and percentage of acetonitrile as organic modifier. Monitored response was a composite quality response (Q*) which balanced conflicting normalized responses, such as resolution and migration time. Optimal separation of the four studied compounds was achieved in less than 6.5 min, using an electrolyte of 60 mM borate buffer with no organic modifier and 25 mM methyl-beta-cyclodextrin concentration adjusted to pH 8.0 at a potential of 30 kV. Detection wavelength and temperature were 197 nm and 20 degrees C respectively. This work means a significant improvement with regard to a previous separation method for these compounds developed in our laboratory.

Different beta-cyclodextrins have been tested as chiral additives in the mobile phase for the chromatographic analysis of chlorthalidone enantiomers in a C18 LiChrospher (125 x 4 mm I.D.) column. The effect on enantioresolution of different parameters was studied: composition of the mobile phase (percentage of organic solvent, type of buffer and pH), mobile phase flow-rate, and type and concentration of beta-cyclodextrin. A 25:75 mixture of methanol and 0.1 M phosphate buffer, pH 4, containing 2% triethylamine (v/v), and 12.5 mM beta-cyclodextrin, at a flow-rate of 0.8 ml/min, was found to be the best option for the resolution of chlorthalidone enantiomers. Under such conditions, linear calibration curves were obtained in the 0.5-20-microg/ml interval using UV detection at 230 nm. The limit of detection for both isomers was 50 ng/ml. The utility of the described assay has been tested by analyzing chlorthalidone in different pharmaceutical preparations. Examples of application to biological samples are also given.

Urodilatin (ANP-(95-126)), a natriuretic peptide in urine, and alpha-hANP (ANP-(99-126)) are crossreactive in the radioimmunoassay of alpha-hANP (ANP-RIA). We therefore developed a method to separate physiological amounts of urodilatin and alpha-hANP in urine by high-performance liquid chromatography (HPLC) followed by ANP-RIA of the separated fractions. We studied urine samples of 10 healthy adult males with a plasma alpha-hANP level of 41 +/- 21 pg/ml (mean +/- SD) and a total urinary ANP-RIA reactivity of 40 +/- 21 pg/ml. In all urine samples we found three peaks of ANP-RIA reactivity, the first one coeluting with synthetic urodilatin, the second one with the retention time of alpha-hANP and a late eluting ANP-RIA-reactive peak, possibly containing degradation products. The ratio of urodilatin/alpha-hANP was 0.77 +/- 0.17.

High-pressure liquid chromatography was used to optimize the resolution of therapeutic agents commonly found in antihypertensive preparations. Seven widely prescribed drugs were investigated. The compounds were chromatographed on reversed-phase octadecyltrichlorosilane (C18) or diphenyldichlorosilane (phenyl) columns, using mixtures of acetonitrile or absolute methanol and distilled water buffered with ammonium acetate, ammonium chloride, or ammonium carbonate. By calculating approximate resolution values, the separation of selected drug mixtures can be predicted.

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.