John K. Baker

Retention index scale for liquid—liquid chromatography

Abstract A retention index scale based on the relative retention of a homologous series of C 3  C 23 2-keto alkanes was used to characterize the chromatographic properties of several drugs with four basic reversed-phase systems each using several mobile phase compositions. The retention index of a given drug was found to be fairly independent of exact composition of the mobile phase. Small changes in the retention index were observed for some drugs following a change in column type and these index changes could be related to changes in selectivity of the solvent—column system.

Identification of drugs by high-pressure liquid chromatography with dual wavelength ultraviolet detection

Abstract Using three solvent-column systems, 101 drugs of forensic interest were characterized by their high-pressure liquid chromatographic relative retention times and by the ratio of their absorbances at 254 and 280 nm. Using relative retention times alone, only 9% of the drugs could be distinguished; while when both the retention times and absorbance ratios were used, 95% of the drugs could be distinguished. The compounds were also characterized by comparisons of their retention times on an adsorption column and a reversed-phase column, however this pair of discriminators were less useful than the former techniques.

Effects of propranolol and a number of its analogues on sodium channels

To assess the relative contributions that the sodium channel blocking activity of propranolol may play in a variety of its therapeutic applications, its effects were examined in vitro with a sodium channel specific 22Na+ uptake system, using rat brain membranes. Propranolol inhibited 22Na+ uptake in the rat brain membrane preparation by acting as a competitive inhibitor of the binding of the sodium channel opening agent veratridine, with an IC50 for this action of 6.5 microM. This is approximately one order of magnitude higher in concentration than that necessary for expression of the beta-adrenergic antagonism of propranolol. The binding of propranolol and its action to block sodium channels were demonstrably different from those of the neurotoxins tetrodotoxin and saxitoxin. Propranolol had effects on sodium channels that are similar, although not identical to those of the local anesthetics procaine and lidocaine. The concentrations of propranolol and a number of its analogues which produced 50% inhibition of 22Na+ uptake (IC50 values ranging from 4 to greater than 100 microM) were similar to the concentrations of these same analogues which were required to produce negative inotropic and antiarrythmic effects (ED40) on isolated rabbit atria [D. O. Rauls and J. K. Baker, J. med. Chem, 22, 81 (1979)]. These effects showed correlations of 0.945 and 0.936, respectively, with the 22Na+ uptake inhibition. It is concluded from this information that a substantial proportion of the negative inotropic and antiarrythmic effects of propranolol is due to its action on sodium channels.

One-Dimensional and Two-Dimensional 1H- and 13C-Nuclear Magnetic Resonance (NMR) Analysis of Vitamin E Raw Materials or Analytical Reference Standards

Two-dimensional spectral analysis (COSY, HETCOR) was utilized to make the complete 13C- and 1H-NMR assignments for α-, β-, -γ-, and δ-tocopherol as well as for the acetate and succinate esters of α-tocopherol. 13C-NMR was found to be especially useful in distinguishing between the various tocopherols and distinguishing between the d-isomer and the d,l-racemic mixture. HETCOR spectra were also found to be useful for the qualitative identification of mixtures of the tocopherols and sesame oil. Using a procedure designed to minimize errors arising from spin relaxation and nuclear Overhauser effects, 13C-NMR peak integrals were used to quantitate α-tocopherol and δ-tocopherol in the presence of sesame oil using benzoic acid as the standard for calibration of the quantitation. The NMR results were compared to a capillary column gas chromatographic analysis of the individual α-tocopherol and δ-tocopherol reference materials.

Metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mouse liver preparations

Using a mouse liver microsomal preparation, it was found that the heterocyclic ring system of MPTP underwent an initial alpha-oxidation to give chemically reactive metabolites that may be associated with the induction of Parkinsonism by MPTP. Subsequent oxidative metabolic transformations of these intermediates were found to give a lactam metabolite and a pyridone metabolite that potentially may interact with the neurotransmitter system.

Laboratory to Laboratory Reproducibility of High Performance Liquid Chromatographic Retention Indices

Abstract Using seven typical drugs, the intra-and inter-laboratory reproducibility of the measured HPLC retention indices, relative retention times, and adjusted relative retention times using the same mobile phase and various reversed-phase C-18 columns were determined. Within a given laboratory, the respective relative standard deviations were ± 0.99%, ± 1.78%, and ± 2.63%. Between laboratories, the respective relative standard deviations were found to be ± 12.6%, ± 30.2%, and ± 34.8%. These results indicated that the HPLC retention index scale may be more useful in comparing data between laboratories.

Decomposition of Arteether in Simulated Stomach Acid Yielding Compounds Retaining Antimalarial Activity

In simulated stomach acid (aqueous 0.01 M HC1, 37°C) β-arteether decomposed (half-life, 441 ± 17 min) to dihydroartemisinin, which subsequently rearranged to a new compound (1) having an endoperoxide group and an aldehyde group. The in vitro antimalarial activity of dihydroartemisinin is similar to that of β-arteether, whereas compound 1 had approximately 1/10th the activity of β-arteether. Compound 1 was prepared in sufficient quantities to afford samples for biological evaluation and a complete chemical characterization with 1H- and 13C-NMR and mass spectrometry. While β-arteether would be somewhat unstable in the stomach, if the drug were administered on an empty stomach (emptying time, ≈30 min) as a suspension or tablet, sufficient quantities of intact arteether may reach the small intestines, where it would be stable and readily absorbed. Its decomposition products, dihydroartemisinin and 1, may also contribute to the antimalarial activity of the administered drug following oral administration.

Structure elucidation and thermospray high-performance liquid chromatography/mass spectroscopy (HPLC/MS) of the microbial and mammalian metabolites of the antimalarial arteether

Microbial metabolism studies of the antimalarial drug arteether (1) have shown that arteether is metabolized to six new metabolites in addition to those previously reported (3). Large-scale fermentations with Cunninghamella elegans (ATCC 9245) and Streptomyces lavendulae (L-105) have resulted in the characterization of these metabolites primarily by two-dimensional nuclear magnetic resonance (2D-NMR) methods as 9β-hydroxyarteether (2), a ring rearrangement metabolite (3), 3α-hydroxy-11-epi-deoxydihydroartemisinin (4), 9α-hydroxyarteether (5), 2α-hydroxyarteether (6), and 14-hydroxyarteether (7). Thermospray mass spectroscopy/high-performance liquid chromatographic analyses have shown that four of these metabolites (2, 5, 6, 7) are also present in rat liver microsome preparations.

High-performance liquid chromatographic analysis of the metabolism of primaquine and the identification of a new mammalian metabolite.

Using rats that had been dosed with 20 mg/kg of primaquine diphosphate (11.4 mg/kg free base), it was found that the drug underwent a metabolic oxidative deamination to give 8-(3-carboxy-1-methylpropylamino)-6-methoxyquinoline. The presence of this new mammalian metabolite was verified using high-performance liquid chromatographic, gas chromatographic, and mass spectral methods. A quantitative high-performance liquid chromatographic method for the determination of primaquine and the carboxylic acid metabolite in plasma using only 50 microliters of whole blood from the rat was developed and the method could be used to detect levels as low as 0.05 microgram/ml of the metabolite. Following intravenous administration of the drug, it was found that the plasma levels of primaquine fell very rapidly and after 30 min, the levels of the metabolite were much higher than those of primaquine.

Determination of Ascorbic Acid and Dehydroascorbic Acid in Blood Plasma Samples

Abstract Following the stabilization of the plasma samples with HClO4 and EDTA, the samples could be directly analyzed by HPLC using electrochemical detection and reversed-phase columns. The accuracy and precision of the method was evaluated using plasma samples spiked with ascorbic acid (10 μg/ml) and the results were also compared to the classical colorimetric procedure. Dehydroascorbic (5 μg/ml) was determined in plasma samples using UV detection following derivatization at room temperature for 45 minutes with o-phenylenediamine.