Springer Science and Business Media LLC

A sensitive high performance liquid chromatographic method has been developed for the determination of cinnarizine in human plasma. Cinnarizine and clocinizine (internal standard) were extracted from acidified plasma (pH 4.7) into carbon tetrachloride and the organic layer was evaporated. The products were separated on a Microspher C18 (3 μm) column, using a mixture of 0.04 % triethylamine in 0.01 M ammonium dihydrogen phosphate (NH4H2PO4), pH adjusted to 4.2 with orthophosphoric acid (H3PO4), and acetonitrile (20∶80, v/v) as mobile phase, at a flow rate of 1 ml/min at 40°C. Fluorescence detection (λex = 245 nm, λem = 310 nm) was used; the detection limit was 0.5 ng/ml under the conditions used, and the calibration curve linear in the concentration range evaluated (1–60 ng/ml). The assay has been used to measure cinnarizine concentrations in plasma after oral administration to volunteers.

The experimentally known dependence in RP-HPLC of the retention factor k′ on octanol/water partition coefficient (K ow) has been examined based on solvophobic theory. The result showed that the dependence provides a means for the evaluation of phase ratio (Φ) of RP-HPLC columns, and of the equilibrium constant for a given compound and mobile phase. Using this theory, the phase ratio was evaluated for a set of seven different C18 columns (five having fully porous particles and two core–shell particles), and the equilibrium constants were calculated for four homologous series of compounds in two mobile phase systems. One mobile phase was methanol/aqueous solution of 0.1% H3PO4, and the other was acetonitrile/aqueous solution of 0.1% H3PO4. Besides providing the values for Φ for the evaluated columns, the results of the study indicated that for a specific composition of the mobile phase and for a given compound displaying only hydrophobic interactions, the equilibrium constant K(X) for different C-18 columns is basically the same. The data were further used to provide guidance in the selection of a chromatographic column for a specific separation based on K ow values and chemical structure of the analytes. The study indicated that the separation of compounds with identical polar groups (or no polar groups) and with very close values for the K ow cannot be achieved based only on hydrophobic interactions that dominate the separation on RP-type columns. Only column that displays polar interactions may provide a solution to such separations. For hydrocarbons with close K ow values, the separation cannot be achieved even on columns with some polarity. On the other hand, even compounds with equal K ow values, but with different functionalities can be separated on RP-HPLC columns without involving polar interactions. The compounds with different K ow values are expected to be easily separated on RP-HPLC columns.

The feasibility of reversed-phase high-performance liquid chromatography for the separation of several metal complexes ofmeso-tetrakis(p-tolyl)porphine (TTP) is described. A combination of an octadecyl-bonded stationary phase with a non-aqueous polar mobile phase, such as an acetone-acetonitrile mixture, has proved effective for the separation. Thus, the TTP complexes of Mg, VO, Ni, Cu, Zn, and Pd and also TTP free acid were successfully separated in about 10min on a Li-Chrosorb RP-18 column (7μm, 250×4mm i.d.) with a 70∶30 (vol/vol) mixture of acetone and acetonitrile at a flow-rate of 1 mlmin−1.

Commercial monolithic columns were evcluated as extraction supports for the analysis of various drugs and their metabolites from plasma. For this purpose, a precolumn packed with a monolithic phase was used as extraction and enrichment support and integrated in a column-switching system for the direct and simultaneous analysis of fluoxetine and norfluoxetine, methadone and EDDP, flunitrazepam and norflunitrazepom in humon plasma. In this configuration, plasma samples were directly injected onto a Chromolith Flash precolumn (25×4.6 mm I.D.) coupled to a microbore analytical column packed with a conventional reversed-phase malerial. Detection was by an electrospray-ionization mass spectrometer (ESl-MS). Total analysis time, including extraction and separation of the six analytes, was <10 min. For all the investigated compounds, the limits of quantitation (LOQ) were estimated in the ng.mL1 range for an injection volume of 50 μL. The method was also applied to a real clinical case to demonstrate its applicability.

A new series of polystyrene-divinyl benzene PS/DVB-based microparticulate packings for size-exclusion chromatography [SEC] is presented. These gel-based packings are characterized by mechanical stability, minimal interaction with solutes and ability to handle a wide variety and size range of polymers and low relative molecular mass samples. Combinations of different pore sizes are discussed and separations of standard polymers and commercial products are shown.

The influence of the alkyl chain length on methylene selectivity has been examined, both in reversed-phase liquid chromatography and liquid-liquid extraction. While log α values remained constant in liquid-liquid extraction, the reversed-phase results demonstrated an increase in selectivity with increasing chain length of the bonded phase.

Based on the linear relationship between large sample injection time during hydrodynamic injection and migration time in non-stacking runs, a new method is proposed to measure average hydrodynamic velocity in capillary zone electrophoresis (CZE). Only the migration times for large injections need to be measured in this new approach, so the method is simple. Additionally, a modified Poiseuille equation is proposed to eliminate the deviation involved in Poiseuille’s equation. The average hydrodynamic velocity values determined by our method generally compare favorably with those determined by the modified Poiseuille equation.

A bioanalytical method has been developed and validated for determination of pregabalin in human plasma. The analytical method consists in the precipitation of plasma sample with trichloro acetic acid (20% v/v solution in water), followed by the determination of pregabalin by an LC-MS-MS method using gabapentin as internal standard. Separation was achieved on a Gemini C18 50 mm × 2.0 mm (3 μm) column with an isocratic mobile phase consisting of methanol–water (98:2, v/v) with 0.5% v/v formic acid. Protonated ions formed by a turbo ionspray in positive mode were used to detect analyte and internal standard. The MS-MS detection was by monitoring the fragmentation of 160.2→55.1 (m/z) for pregabalin and 172.2→67.1 (m/z) for gabapentin on a triple quadrupole mass spectrometer. The assay was calibrated over the range 0.1–15.0 μg mL−1 with correlation coefficient of 0.9998. Validation data showed intra-batch (n = 6) CV% ≤ 6.89 and RE (%) between −4.17 and +3.08 and inter-batch (n = 18) CV% < 9.09 and RE (%) between −3.0 and +10.00. Mean extraction recovery were 80.45–89.12% for three QC samples and 87.56% for IS. Plasma samples were stable for three freeze–thaw cycles, or 24 h ambient storage, or 1 and 3 months storage at −20 °C. Processed sample (ready for injection) were stable up to 72 h at autosampler (4 °C). This method has been used for analyzing plasma samples from a bioequivalence study with 18 volunteers.

A differentiating time printer (DTP) apparatus has been constructed from standard laboratory equipment by connecting a differentiating CR circuit via an operational amplifier to an electronic temperature regulator that triggers the print out of a line frequency driven register. The electronic resolution is 0.1 s, giving a relative standard deviation σrel, which is determined by the stability of the line frequency at sufficiently long retention times. In Lund this corresponds to a best σrel = 0.02% and to a mean σrel = 0.03%. For late and broad peaks in real GC the noise caused the print-out of extra time values, especially with fronting peaks from ordinary 1/8″ columns. However, a comparison with the simultaneously recorded chromatograms was generally sufficient to pick out the proper retention times. For large sample sizes the corrected retention time t′ increased with peak height, the later the peak in the chromatogram. The In t n+1 ′ /t n ′ values from a series of n-paraffins showed good linear correlations with peak height hn+1 and peak area An+1, giving standard deviations around the line approaching that expected from σrel. The accuracy of the system was mainly determined by the effect of a non-zero triggering level on the derived signal and by the time displacement expected from a large time constant. A rough estimate of the time error was made by simulating GC peaks with a cosine wave generator.