Rapid hydroxylation of methtryptoline (1-methyltetrahydro-β-carboline) in rat: Identification of metabolites by chiral gas chromatography-mass spectrometry

Summary Racemic methtryptoline (1-methyltetrahydro--carboline) and 5-hydroxymethtryptoline-9-carboxylic acid (6-hydroxy-1-methyltetrahydro--carboline-1-carboxylic acid) were administered intraperitoneally to rats and the components of their urine was subsequently investigated by chiral gas chromatography-mass spectrometry. Methtryptoline rapidly became hydroxylated in the 5- and 6-position and excreted in urine. There was about a ninefold predominance of the S(–) enantiomer over the other in the 5-hydroxylated species, while the 6-hydroxylation produced a small excess of the R(+) enantiomer. About 75% of the injected dose of methtryptoline was recovered in the urine as 5- and 6-hydroxylated compounds during the first 24 h period, demonstrating that hydroxylation represents the major metabolic pathway. Treatment with 6-hydroxymethtryptoline-9-carboxylic acid led to a fivefold increase in the urinary excretion of 5-hydroxymethtryptoline during the first 24 h period with a predominance of the S(–)-enantiomer, indicating a much smaller conversion rate than from methtryptoline. It was concluded that hydroxylation of methtryptoline is a likely pathway for the natural formation of 5-hydroxymethtryptoline.     

Determination of free fractions of tricyclic antidepressants

Summary The plasma protein binding of amitriptyline, imipramine, clomipramine, and their primary demethylated metabolites were studied by means of a method combining dialysis and gas chromatography. Equilibrium in dialysis of serum containing amitriptyline and its metabolite nortriptyline was attained in about 0.5 h with the drug dissolved in the serum compartment, and in about 2 h with the drug passing from the buffer to the serum compartment.The calculation of free fractions was influenced by variations with dialysis time in the volumes of serum and buffer. Increase of pH in serum increased the protein binding of the weakly basic drugs studied, and made the Donnan distribution effects more pronounced. At pH 7.4, the Donnan effect was negligible.Binding parameters for the 6 tricyclic antidepressant substances studied were estimated for the binding to ₁-acid glycoprotein and for total binding in serum. For ₁-acid glycoprotein, the k-values ranged from 1·10⁵ to 8·10⁵ M^–1, and for pooled serum from 0.4·10⁵ to 8·10⁵ M^–1. The determined number of binding sites on the ₁-acid glycoprotein was, on average 0.87 for the 6 substances. In serum, the binding capacity was 2–14 times the concentration of ₁-acid glycoprotein.     

Dissolution test for felodipine tablets using chemical oxidation in situ to maintain 'sink conditions'

A test model is described for the determination of the dissolution rate of the vasodilator, felodipine, a derivative of dihydropyridine that is practically insoluble in water. ‘Sink conditions’ are maintained by means of an oxidizing agent, ceric sulphate, which reacts rapidly with dissolved drug molecules in the dissolution fluid. A pyridine derivative is formed quantitatively in the oxidation reaction. The amount of dissolved felodipine is calculated from the concentration of the pyridine derivative, as determined by reversed-phase liquid chromatography. Dissolution rates depend on the concentration of the oxidizing agent so that high concentrations accelerate dissolution. The dissolution test suggested for 25-mg felodipine tablets is performed in 500 ml fluid that contains 5 mM ceric sulphate in 0.12 M sulphuric acid. The test is performed on single tablets with USP paddle equipment. Dissolution rates for nine different tablet compositions are correlated to such bioavailability parameters as maximum plasma concentration and total area under the plasma concentration—time curve. Interferences and limitations of the method are discussed.     

Stability-indicating determination of adrenaline in injections containing procaine

Adrenaline was determined in injections containing procaine in a 1000-fold excess by reversed-phase high-performance liquid chromatography using UV detection at 205 nm and aqueous sulphuric acid (100 μmol/l) as eluent. The relative standard deviation was 2.1%, and the method was selective in the presence of adrenaline degradation products. Changes of the capacity factor with pH and ionic strength of the eluent were studied, and a simple model is suggested to explain the retention data.     

New detection schemes in liquid chromatography

A micropolarimeter interfaced to a liquid chromatograph is shown to be suitable for selective monitoring of the optically-active components in complex samples. When an optically-active eluent is used, indirect determination of even optically-inactive materials is possible, down to the level of 10 ng of an injected component. If a second chromatogram is obtained using the racemic analogue of the optically-active eluent, quantitation can be achieved without standards and without prior analyte identification. This concept is also applicable to the refractive index detector, the absorption detector and the conductivity detector in the special case of ion chromatography, and the ultrasonic detector in gas chromatography.     

Enrichment techniques and trace analysis with microbore columns in liquid chromatography

The advantages of microbore columns for trace analysis by liquid chromatography are identified, with reference to on-column enrichment techniques performed on analytical micro-columns. The selectivity and high sensitivity of the amperometric detector are utilized in combination with a microbore column for a number of pharmaceutical and bioanalytical analyses, including phenothiazines, parabens, sulphonamides, catecholamines, tetracyclines, vitamins, amino acids and dipeptides.     

The combined use of high-performance liquid chromatography and radioimmunoassay for the bioanalysis of nicomorphine and its metabolites

Methods have been developed for the determination of nicomorphine using reversed-phase HPLC with UV detection; for the simultaneous assay of morphine and mononicotinoylmorphine by a coupled normal-phase HPLC-radioimmunoassay method; and for conjugates of morphine and mononicotinoylmorphine by radioimmunoassay. The methods have been evaluated and applied to a pharmacokinetic study of nicomorphine administered intramuscularly.     

HPLC analysis of 16 compounds from Artemisia ordosica

Objective: To establish a high-performance liquid chromatographic method (HPLC) for the simultaneous determination of sixteen compounds from Artemisia ordosica. Methods: HPLC was used to analyze 16 quality indicators of A. ordosica. The HPLC conditions were as follows: Agilent Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) with acetonitrile (A)-water (B) as mobile phase, gradient elution: 0?10 min, 75%?65% B; 10?30 min, 65%?35 % B; and finally 30?40 min, 35%?15% B. The flow rate was 1.0 mL/min, the column temperature was 40 °C, the injection volume was 10 μL, and monitored by absorbance at 285 nm for compounds 1?10, 12 and 225 nm for compounds 11, 13?16. Results: Under the selected experimental chromatographic conditions, compounds 1?16 showed good linearity (r > 0.9993) in a wide concentration range. Their average recoveries were 99.50%, 95.38%, 97.75%, 96.00%, 98.20%, 97.50%, 95.50%, 99.33%, 96.75%, 96.50%, 98.50%, 97.83%, 99.20%, 95.33%, 97.33% and 96.30%, respectively, and the RSD were 1.99%, 1.81%, 1.63%, 1.98%, 1.67%, 1.92%, 1.74%, 1.67%, 1.90%, 1.72%, 1.88%, 1.83%, 1.79%, 1.76%, 1.81% and 1.96%, respectively. Conclusion: Based on the results of the HPLC analysis, it was concluded that p-hydroxycinnamic acid (1), O-hydroxycinnamic acid (2), coniferyl alcohol (5), 5,4''-dihydroxy-7,3''-dimethoxyflavanone (8), 5,4''-dihydroxy-7-methoxyflavanone (9), 5-hydroxy-7,4''-dimethoxyflavanone (12), dehydrofalcarindiol (13), arteordoyn A (14), dehydrofalcarinol (15) and capillarin (16) are best suited for the role of quality indicators of A. ordosica grown in different ecological environments.     

Directional screening and identification of potential cytotoxic components from Achnatherum inebrians by a combination of surface palsmon resonance and chromatography

Objective: To establish a method for directional screening of the cytotoxic components from the medicinal herb of Achnatherum inebrians by a combination of surface plasmon resonance (SPR) biosensor and chromatographic isolation technology. Methods: Under the guidance of bioactive assessment based on binding abilities between objects and the α-Mannosidase (α-Man) target, the active components from different solvents extracts, different polar extraction parts and fractions were screened orderly and directionally using SPR. Components with a high binding ability to α-Man can be precisely oriented in a narrower fractions range and are easy to isolate. Three human cancer cells were used to evaluate the cytotoxic activity of component with the highest affinity to α-Man. Results: Eight compounds were isolated and identificated from A. inebrians for the first time. Deoxyvasicinone possessed the highest affinity to α-Man among them. Moreover, deoxyvasicinone showed good effects on inhibited proliferation of human hepatoma cells HepG2 (IC50 = 5.7 μmol/L), human breast cancer cells MCF7 (IC50 = 7.21 μmol/L) and human lung cancer cells HCC827 (IC50 = 0.75 μmol/L), respectively. In particular, its inhibitory effect on HCC827 was stronger than the positive drug gefitinib (IC50 = 1.65 μmol/L). Conclusion: A comprehensive strategy of directional screening potential cytotoxic components from herb based on biomolecular interaction and chromatography was established. Deoxyvasicinone as an effective anti-cancer component was initially isolated from A. inebrians. It is expected that this screening strategy could provide new perspectives for rapid screening and identification of active components from natural plants with the complex matrix.