Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Propafenone is a new class 1C antiarrhythmic agent with additional calcium antagonistic and beta-blocking activities. Clinically it is effective in the treatment of supraventricular and ventricular tachycardia, atrial and ventricular fibrillation, ventricular premature contractions and for the management of Wolf-Parkinson-White syndrome. In North America it is still an investigational drug. Propafenone is a chiral drug and is used clinically in the racemic form. The enantiomers of numerous chiral drugs have been shown to differ in their disposition kinetics in the body due to their stereoselective pharmacokinetics and/or pharmacodynamic properties. Two enantiomers are thus often considered as two different entities. The relative antiarrhythmic activities of individual enantiomers of propafenone have not been studied, nor their pharmacokinetic parameters have been elucidated. In order to study the possible enantioselective role of propafenone in the body, a stereoselective assay method would be required. The present study describes the development of a sensitive and stereoselective chromatographic assay method for the simultaneous determination of the two enantiomers of propafenone in human plasma. Attempts for direct separation of the enantiomers of propafenone included several GLC and HPLC chiral stationary phases. The chiral stationary phases were a Chirasil-Valʳ GLC stationary phase, a Pirkle 2,4 dinitro-(D)-phenylglycine HPLC stationary phase and a β-cyclodextrin HPLC stationary phase. Unfortunately, these did not resolve the enantiomers of propafenone. Formation of the diastereomers with R(+)-⍺-methyl benzyl isocyanate and racemic propafenone were partially resolved on a reverse phase HPLC using a 5 u, 25 x 0.45 cm i.d. ODS column and methanol/water (70:30) as the mobile phase. However, due to the long retention time (42 min), incomplete resolution (RS=1.15) and poor sensitivity for detection (500 ng of each enantiomer injected) this method was not deemed suitable for the pharmacokinetic studies planned, since the therapeutic plasma concentration range of propafenone is 64-1044 ng/mL. The second chiral derivatizing reagent, 2,3,4,6-tetra-0-acetyl-β-D-glucopyranosylisothiocyanate (GITC), was synthesized in our laboratory. This reagent gave better resolution of the enantiomers (RS=1.4) within 15 minutes with enhanced sensitivity for detection (150 ng of each enantiomer injected). To further optimize the limit of detection for future pharmacokinetic studies of propafenone, R(-)-1 -(naphthyl) ethylisocyanate, a chiral derivatizing agent, was employed. This reagent reacted with racemic propafenone and permitted the resolution of both enantiomers within 24 minutes (R5=l.25) and the minimum level of detection was 100 ng (at the detector) for each enantiomer of propafenone. Using this method, linearity was established over the concentration range, 125-1000 ng for each enantiomer (injected) with a coefficient of determination (r²) of greater than 0.99. Reproducibility and precision of this assay method was obtained with an average coefficient of variability of 4.5% for the R(-) enantiomer and 7.2% for S(+) enantiomer at concentrations of 125-1000 ng/mL. Below the lower quantity, the NEIC-propafenone reaction virtually stopped at the conditions set for derivatization. A similar lack of reactivity at low concentrations was also observed with the GITC-propafenone reaction. The absence of an autocatalysing effect of propafenone at lower nanogram levels, as well as two possible conformational forms of propafenone were also investigated. The existence of two conformational isomers of propafenone, due to intramolecular hydrogen bonding in aprotic solvents, was chromatographically verified. In addition, chromatographic separation of all the proposed conformers was obtained, indicating that enantiomeric separation and quantitation of propafenone enantiomers as their urea derivatives is substantially hindered. To eliminate hydrogen bonding interactions, the carbonyl group of propafenone was blocked with dansylhydrazine and subsequently derivatized with the chiral R(-)NEIC reagent. The HPLC resolution (RS=1.35) of this dual derivative was better than that using the R(-) NEIC reagent alone, and the minimum level of detection was 2.5 ng for each enantiomer. Unfortunately, this procedure still did not provide adequate assay precision and accuracy at the lower levels required for single dose pharmacokinetic studies. / Pharmaceutical Sciences, Faculty of / Graduate

The physical and chemical nature of chemically bonded phases used in high performance liquid chromatography have been studied. These bonded phases were characterized by a variety of chromotographic and non-chromatographic experiments. The non-chromatographic techniques included ¹³C NMR and batch extraction methods. The role played by the bonded phase as well as the mobile phase in determining the retention characteristics of the "stationary phase" were determined. The retention of solute molecules on bonded phases was found to be a function of the chain length of the bonded phase, the chemical nature of the bonded molecule, and the type of organic modifier used in the mobile phase. The energetics of the solute-stationary phase interactions was determined by the differential enthalpy and was found to be indicative of a partitioning process between two liquid phases. The retention process was also affected by the surface coverage of the bonded molecule. Optimum retention and separation characteristics were obtained with a hydrocarbon bonded phase of high surface coverage when used with a mobile phase containing a very polar organic modifier. The efficiency of these bonded phases was found to be independent of chain length as well as surface coverage of the bonded molecule. Some bonded phases which have specific functionalities incorporated into the bonded molecule are not true reversed phases. The selectivity of the bonded phases towards polar solute molecules was found to be affected by the type of organic modifier used in the mobile phase. The major accomplishment of this work shows that the stationary phase consists of the bonded molecule as well as trapped mobile phase. The composition of this ternary mixture is a function of the type and amount of bonded material and the type and amount of organic modifier used in the mobile phase.

Dietary cyclopropenoid fatty acids (CPFA) have long been known to cause pronounced physiological disorders in both farm and laboratory animals. Past work has shown CPFA to be a powerful promoter of carcinogenesis in trout, rats and mice. Sterculic and malvalic acids (CPFA's) are found in seed lipids of plants from the order Malvales. Two members of this order are cotton and kapok, both of which are used extensively as cooking oils for human consumption. Present chemical and instrumental methods of analysis for CPFA are effective only at CPFA levels above 0.1%, and accurate only at levels above 1%. A more sensitive method of analysis was developed exploiting recent technological advances in glass capillary gas chromatography (GCGC). By the use of cold on-column injection, and positioning of the column at the base of the detector flame, this method eliminates two problem areas found in other GC methods. The other common component involved in CPFA decomposition, during GC analysis, is the column. Vitrious silica columns with an inert stationary phase, SE-30, were shown to separate the highly reactive CPFA without decomposition. Proof of stability during analysis was obtained by changing the variables of relative time on column and column temperature. This method not only allows individual determination of sterculic and malvalic acid concentrations, it is rapid, accurate (to the 70 parts per million range), and is superior to other instrumental and chemical methods. The CPFA concentration for okra, hollyhock, cheese weed, seashore mallow, kapok, and white cap cottonseed oil are as follows: 0.3% to 0.92%, 0.33%, 2.6%, 2.6%, 12.8%, and 75ppm, respectively. The two different values for okra were found because seeds from two different growing seasons were analyzed. No CPFA could be detected in Diet Imperial Margarine, raw cocoa beans, cocoa butter or Lucca's winterized cottonseed oil. / Graduation date: 1987

The use of gas chromatography for the separation, comparison, and subsequent identification of flavor volatiles from food products has proven highly successful in recent years. The development of various techniques for the concentration of the volatiles before gas chromatographic analysis has greatly extended the use of this important analytical tool. The injection of vapors directly into the chromatograph without prior concentration is the simplest method and has been used successfully on many food products. However, the use of this technique on the volatiles of fishery products has met with limited success. The complexity and nature of the flavor compounds found in fishery products have required the use of highly sensitive instruments and columns with very efficient separation power. The purpose of this investigation was to develop a method for the separation and comparison of volatiles from fishery products by this direct vapor injection technique. Preliminary investigations showed that a nine foot column of diisodecyl phthalate on 80/100 mesh, methanoic KOH treated, celite 545, operated isothermally at 35°C, would give satisfactory separation of one to three ml samples of volatiles from heated fishery products. The technique was not, however, sensitive enough to allow direct sampling of cold products unless they were highly spoiled or autoxidized. This investigation showed that direct vapor injection, using the column and conditions described, will show differences between size and number of peaks in heated fresh, oxidized, and spoiled fishery products. Several peaks in autoxidizing menhaden oil were shown to increase with hours of oxidation and a peak with the same retention time as trimethylamine was observed in the chromatograms of spoiled fish. The direct injection technique did not show large differences between fresh dover sole, rockfish, oysters, or beef. Tentative identification of various peaks from the chromatograms of oxidized salmon oil was attempted by comparison of retention data to known compounds and by functional group analysis by the method of Hoff and Feit (34). In this manner the possible existance of C₁ to C₇ alkanals, 2-hexen-1-al, methane, heptane, ethanol, butanol, and acetone was shown. The methods of tentative identification used were preliminary in nature and confirming tests would be necessary before positive identifications could be made. A comparison of chromatograms from fish, oysters, beef, and fish oils showed that several similar peaks appear in every case. These peaks were found at retention times of 0.71, 0.87, 1.42, 2.21, 2.83, 3.62, 5.20, and 5.51 minutes. / Graduation date: 1964

No description available.

No description available.

The use of a new styrene-divinylbenzene copolymer bead, Showdex Polymerpak D-814, as a stationary phase in high-performance liquid chromatography is investigated. Unlike conventional silica-based stationary phases, copolymer beads may be used with both aqueous and organic mobile phases. The effect of the mobile phase on solute retention with the copolymer beads is described. Although the copolymer beads exhibit characteristics of both solid and liquid stationary phases, it is shown that the beads act primarily as a liquid when used with the mobile phases chosen for this work. It is further shown that solute retention on the beads results from dispersion interactions between the solute and the stationary phase. Batch extraction distribution constants are determined to confirm the validity of the distribution model proposed. The chromatographic behavior of a variety of aromatic compounds is described. These range from polycyclic aromatic hydrocarbons to substituted benzenes to phthalate esters. A quantitative measure of the effect of a substituent group on retention is developed from an analysis of the experimental results. This measure is then used to successfully predict the retention behavior of some disubstituted benzene compounds. Some model separations are developed to illustrate the usefulness of this measure. Finally, the implications of this work for gradient elution chromatography are discussed.

No description available.

No description available.