Evidences for temperature-dependent mechanism of host-guest complexation

Summary High-performance liquid chromatography and ultraviolet spectroscopy methods were applied to the studies on the influence of temperature on the complexation of β-cyclodextrin with naphthalene and its derivatives. The strong nonlinearity of Van't Hoff plots suggests, that the retention mechanism of hydrocarbons investigated might be different in high and low temperature region. The total lack of correlation (r=−0.230) between chromatographic data (capacity factors ratio:k _PAH/k _PAH×CD) and spectrophotometric data (ΔA) at high temperature (60°) as well as a significant correlation (r=0.922) at subambient temperature (15°C) suggest, that the inclusion mechanism starts to be important at low temperature region and the predominant mechanism for chromatographic retention is the formation of an inclusion complexes in the mobile phase.     

Trace analysis of selected benzidine and diaminodiphenylmethane derivates in urine by means of liquid chromatography using precolumn sample preconcentration,UV and electrochemical detection

Summary For the title compounds 4,4-DADPM, MOCA, 3,3-DCB, 4-ADP and 4-ADPA listing on the EPA priority pollutant list, an analytical practicable, reliable, reproducible and sensitive procedure is required. Therefore a new method has been developed for the routine determination of these toxic aromatic amines in urine at the ppb level. The quantitative determination of amines is a suitable procedure of occupationally exposed persons. Urine sample preparation is done using simple liquid-liquid extraction followed by a precolumn enrichment (PRP₁-material; Hamilton). Breakthrough measurements were done using an enrichment column packed with PRP₁ material. The capacities of the studied amines ranged from 21.9mg/g to 96.6mg/g, while influent concentrations differed from 28.3mg/l to 332.0mg/l. The advantages of electrochemical detection regarding to selectivity and sensitivity are clearly indicated in this paper. Separation has been achieved applying reversed-phase-high-performance-liquid chromatography (LiChrosorb RP 18/5m) followed by electrochemical or UV-detection. The detection limits employing an electrochemical detector at a potential of 1 V range from 2.2ng to 12.1ng. UV detection at 254 nm and 280 nm is about 10–100 times less sensitive. Recoveries from spiked water samples at the 5ppb levels were 75% to 96% respectively. The standard deviation of the developed procedure varies from 5.3% to 14%. Day-to-day repeatability is good.     

Liquid Chromatographic Analysis of Arabinosylcytosin and 1-β-D-Arabinofuranosyluracil in Treatment with Cyclophosphamide and Pirarubicin

A reversed-phase liquid chromatographic (RPLC) method has been developed for determination of arabinosylcytosin (Ara-C) and its metabolite 1-β-D-arabinofuranosyluracil (Ara-U) on a 250 mm × 4.6 mm i.d., 5 μm particle, Diamonsil C₁₈ column. The mobile phase was a mixture of 5% methanol and 95% 10 mmol L⁻¹ phosphate buffer adjusted to pH 5.5. The flow-rate was 1.0 mL min⁻¹ and the injection volume 20 μL. Eluting compounds were detected at 270 nm by use of an ultraviolet detector. Under these LC conditions cyclophosphamide (CTX) and pirarubicin (THP), two other medicines given with Ara-C in clinical treatment, do not interfere with measurement of Ara-C and Ara-U. Individual calibration plots of peak area against concentration generated from analysis of standard solutions were used to calculate the concentrations of Ara-C and Ara-U in sample solutions. The calibration plot was linear in the range 2.5–100 μg mL⁻¹, the average recovery of Ara-C and Ara-U was more than 98% (RSD < 2.5%), and between-day and within-day precision, expressed as relative standard deviation (RSD), were below 4.0%. LOQ for both Ara-C and Ara-U was 2 μg mL⁻¹. The method is rapid, simple, accurate and reproducible, and especially useful for application to patient samples.     

Structure of hydroxycinnamic acid derivatives established by high-perfomance liquid chromatography with photodiode-array detection

Summary The present paper describes the development of a method for the differentiation of hydroxycinnamic acid derivatives by HPLC with photodiode-array detection. Furthermore spectral data of the compounds under investigation are given. Whereas p-coumaric acid derivatives are distinguishable from caffeic and ferulic acid derivatives by the positions of their spectrum maxima and their convexity interval value, it is not possible to distinguish between caffeic and ferulic acid derivatives because of overlapping spectrum maxima and convexity interval values.     

Ion chromatographic separation of alkylsulphonic acids with conductivity detection

Summary An ion chromatographic (IC) method has been developed for the separation of alkylsulphonic acid. Two different stationary phases, silica-based and polymer-based ion-exchange resins, were studied using pure ion exchange and/or hydrophobic interaction mechanisms. Correlations between analyte hydrophobicity and eluent polarity were made in order to investigate the possibility of changing the dominant separation mechanism by varying the eluent composition. The alkyl chain lengths of the sulphonic acids analysed ranged from C₁ to C₉. Detection limits in the submicromolar range were obtained by suppressed conductivity detection.     

Simultaneous assay of four stereoisomers of diltiazem hydrochloride. Application to in vitro chiral inversion studies

Summary The simultaneous stereospecific assay of four stereoisomers of diltiazem hydrochloride in bulk drug and aqueous solution was developed using HPLC on a Chiralcel OF column. The four isomers were quantitated with good precision by the internal standard method. The chiral inversion of (+)-cis-diltiazem hydrochloride in vitro, stability of its (2S, 3S) configuration in the solid and aqueous states was examined by HPLC. Chiral inversion of (+)-cis-diltiazem hydrochloride was not observed in the solid state, and its (2S, 3S) configuration was stable to heat, humidity and light. Chiral inversion of (+)-cis-diltiazem hydrochloride (2S, 3S) was observed in aqueous solution under UV, but not in aqueous solution stored at 80°C for 5h nor under visible light for 10 h. The (+)-cis-diltiazem hydrochloride (2S, 3S) epimerized to (+)-trans-diltiazem hydrochloride (2R, 3S) with a half-life of 5h in aqueous solution under UV but the reverse chiral inversion of (+)-trans-diltiazem hydrochloride (2R, 3S) to (+)-cis-diltiazem hydrochloride (2S, 3S) was not observed.     

Development and Validation of a Liquid Chromatographic Method for Determination of Efavirenz in Human Plasma

A simple, rapid, and sensitive high-performance liquid chromatographic method for estimation of efavirenz in human plasma has been developed and validated. Chromatography was performed with C₁₈ analytical column and 50:50 acetonitrile–phosphate buffer (pH 3.5) as mobile phase. Compounds were monitored by UV detection at 247 nm. The retention time for efavirenz was 6.45 min and that for the internal standard, nelfinavir, was 2.042 min. Response was a linear over the concentration range of 0.1 μg–10 μg mL⁻¹ in human plasma. The method was simple, specific, precise and accurate and was useful for bioequivalence and pharmacokinetic studies of efavirenz.     

Affinity Chromatography of Insulin with a Heptapeptide Ligand Selected from Phage Display Library

A heptapeptide phage display library was screened with insulin to find its ligands for affinity chromatography. The peptide was synthesized and coupled to EAH Sepharose 4B (5.4 mol mL^–1 bed). Then, insulin chromatography was carried out with mobile phases of different pH values and by the addition of urea and ethylene glycol. It was found that electrostatic interactions were predominant for the affinity binding, and hydrogen bonding might also contribute somewhat to the affinity. Finally, frontal analysis was performed and the dynamic binding capacity of the affinity column for insulin at 50% breakthrough was estimated at 60.6 mg mL^–1 bed, which was about two times higher than the theoretical binding capacity of the monomeric insulin. The result suggests that insulin was bound in dimer state in a stoichiometric relationship with the coupled peptide, indicating the high binding efficiency of the peptide ligand for insulin.     

Sensitive Determination of Felodipine in Human and Dog Plasma by Use of Liquid–Liquid Extraction and LC–ESI–MS–MS

Summary A sensitive and selective liquid chromatographic method coupled with electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) has been developed for quantification of felodipine in human and dog plasma. Compounds were separated on a 2.0 mm × 150 mm, 5.0 m particle, C₈ column with 1 m m ammonium acetate–acetonitrile, 20:80, pH 6.0, as mobile phase at a flow rate of 200 L min^–1. Nifedipine was used as internal standard. Plasma samples were extracted with diethyl ether, the centrifuged upper layer was evaporated, the residue was reconstituted with mobile phase, and the reconstituted samples were injected. The analytical column lasted for at least 1000 injections. By use of multiple reaction monitoring (MRM) mode in MS–MS felodipine and nifedipine were detected without severe interference from the human or dog plasma matrix. Felodipine produced a protonated precursor ion ([M + H]⁺) at m/z 384 and a corresponding product ion at m/z 338. And internal standard (nifedipine) produced a protonated precursor ion ([M + H]⁺) at m/z 347 and a corresponding product ion at m/z 315. Detection of felodipine in human and dog plasma was accurate and precise, with a limit of quantification of 0.05 ng mL^–1. The method has been successfully applied to preliminary pharmacokinetic study of felodipine in human and dog plasma.     

Capillary electrophoresis as an alternative to HPLC for determination of honey flavonoids

Summary The general objective is to provide an alternative methodology based on capillary electrophoresis (CE) to characterize flavonoids from honey and hence determine its botanical origin. The specific objective is to compare the separation of flavonoids by CE with those achieved by HPLC to assess CE as an alternative technique for the determination of honey flavonoids. Fourteen different flavonoids isolated from honey were analysed by MECC and compared to the HPLC separations. It was difficult to find specific experimental conditions to separate all the flavonoids from honey in a single MEKC run. Three chromatographic conditions are optimized and, depending on the flavonoid markers sought in honey, the appropriate detection method should be chosen. Compared to the HPLC results, it is clear that CE could be an alternative technique in honey flavonoids analysis and particularly in the study of its geographical and floral origin.