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A method of high performance liquid chromatographic separation of clausenamide enantiomers with chiral-AGP (α 1 -acid glycoprotein) stationary phases has been established. The absolute configurations of (−)clausenamide and (+)clausenamide are 3 S , 4 R , 5 R , 6 S and 3 R , 4 S , 5 S , 6 R , respectively. The present method has been used to analyze the (−)clausenamide and (+)clausenamide and its analogues such as the major metabolite and synthetic derivatives of clausenamide.

1, 5-Dihydroxy-2, 3-dimethoxy-xanthone (HM-5) is one of the naturally-occurring xanthones of a Tibetan medicinal herb Halenia elliptica. Recently, it has been shown that HM-5 is one of the phase I metabolites of 1-hydroxy-2, 3, 5-trimethoxy-xanthone (HM-1), the major active component of H. elliptica with potent vasorelaxant actions. This study investigated the vasorelaxant effect of HM-5 and its mechanism(s). HM-5 (0.35-21.9 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 4.40+/-1.08 microM. Unlike HM-1, the effect of HM-5 was endothelial-independent such that removal of the endothelium did not affect its vasodilator potency. Nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM) did not affect the vasodilatory effects of HM-5, thus confirming the non-involvement of endothelium related mechanisms. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-5 was inhibited by a potassium channel blocker, TEA (10 mM), and 4-aminopyridine (4-AP, a K(v) blocker; 1 mM) but not by other K+ channel blockers such as iberiotoxin (100 nM), barium chloride (100 microM) and glibenclamide (10 microM). The involvement of Ca2+ channel was studied in artery rings pre-incubated with Ca2+-free buffer (intact endothelium or endothelium-denuded) and primed with 1 microM 5-HT or 60 mM KCl prior to the addition of CaCl2 to elicit contraction. In the 5-HT-primed preparations, HM-5 (34.7 microM) significantly inhibited the CaCl(2)-induced vasoconstriction (89.9% inhibition in intact endothelium artery rings; 83.3% inhibition in endothelium-denuded rings). In the KCl-primed preparations, HM-5 (34.7 microM) produced a 34% inhibition in endothelium-denuded rings. The same concentration of HM-5 inhibited (by 62.3%) the contractile response to 10 microM phorbol 12, 13-diacetate (PDA), a protein kinase C activator, in Ca2+-free solutions. Taken together, this study showed that the mechanisms of the vasorelaxant effects of HM-5 were distinctly different from those of its parent drug HM-1. The vasorelaxant effect of HM-5 was mediated through opening of potassium channel (4-AP) and altering intracellular calcium by partial inhibition of Ca2+ influx through L-type voltage-operated Ca2+ channels and intracellular Ca2+ stores.

1-Hydroxy-2, 3, 5-trimethoxyxanthone (HM-1) is a xanthone isolated from Halenia elliptica, a Tibetan medicinal herb. HM-1 (0.33-42.1 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 1.67+/-0.27 microM. Removal of the endothelium significantly affected the vasodilator potency of HM-1, resulting in 46% decrease in E(max) value. The endothelium-dependent effects of HM-1 was confirmed when its vasorelaxant effect was inhibited after addition of nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (100 microM) or the soluble guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM). Atropine (100 nM), flurbiprofen (10 microM), propranolol (100 microM), pyrilamine (10 microM), cimetidine (10 microM) and SQ22536 (100 microM) had no effect on the vasorelaxant activity of HM-1 indicated the non-involvement of other receptor/enzyme systems. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-1 was unaffected by potassium channel blockers such as tetraethylammonium (10 mM), iberiotoxin (100 nM), barium chloride (100 microM) and 4-aminopyridine (1 mM). The involvement of Ca(2+) channel in 5-HT-primed artery ring preparations incubated with Ca(2+)-free buffer was confirmed when HM-1 (9.93 microM) partially abolished the CaCl(2)-induced vasoconstriction (87% inhibition in intact-endothelium artery rings; 50% inhibition in endothelium-denuded rings). In the KCl-primed preparations incubated with Ca(2+)-free buffer, HM-1 (9.93 microM) produced a 27.3% inhibition in endothelium-denuded rings. HM-1 (3.31-33.1 microM) had minimal relaxant effects (14.4%-20.3%) on the contractile response generated by 10 microM phorbol 12,13-diacetate (PDA) in Ca(2+)-free solutions, suggesting minimal effects on intracellular Ca(2+) mechanisms. These findings suggest the vasodilator action of HM-1 involved both an endothelium-dependent mechanism involving NO and an endothelium-independent mechanism by inhibiting Ca(2+) influx through L-type voltage-operated Ca(2+) channels; a minor contribution to the effects of HM-1 may be related to inhibition of the protein kinase C-mediated release of intracellular Ca(2+) stores.

Supercritical fluid chromatography, using carbon dioxide as the mobile phase and ethanol as a modifier, has been applied to the analysis of products formed in rat liver microsomal metabolism of racemic camazepam, a hypnotic/anxiolytic drug in clinical use. An achiral (amino) column and a chiral (Chiralcel OD-H) column were used. The results suggest that achiral and chiral packed-column supercritical fluid chromatography gives a shorter analysis time and higher selectivity and efficiency than achiral and chiral stationary-phase high-performance liquid chromatography in the analysis of camazepam and its derivatives.

In this study, six major xanthones, isolated and identified from Halenia elliptica were investigated for their vasodilatory actions in isolated rat coronary artery. The xanthones, including 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1), 1-hydroxy-2,3,4,7-tetramethoxy-xanthone (HM-2), 1-hydroxy-2,3,4,5-tetramethoxy-xanthone (HM-3), 1,7-dihydroxy-2,3,4,5-tetramethoxy-xanthone (HM-4), 1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5) and 1,7-dihydroxy-2,3-dimethoxy-xanthone (HM-7) caused vasodilation in the coronary artery pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with EC(50) values ranging from 1.4+/-0.1 microM (HM-1) to 6.6+/-1.4 microM (HM-2). The EC(50) values of the other xanthones were between those of HM-1 and HM-2. Removal of endothelium of the coronary artery led to decreases in the vasorelaxant effects of HM-1, HM-7 but not HM-2, HM-3, HM-4 and HM-5. Our results showed that xanthones isolated from Halenia elliptica are vasoactive substances which exhibit either endothelium-dependent or endothelium-independent mechanisms in rat coronary artery. The potency and mechanism(s) of the vasorelaxant effects of these xanthones may be relevant to the structure-activity differences in the level and the position of the substituent groups with the primary xanthone structure.

To establish a HPLC method for determination two constituents in bark of Paeonia Suffuticosa.The reversed phase HPLC system consisting of an Alltima ODS column (4.6 mm x 150 mm, 5 microm) and a mixture of water-THF-methanol-HAc (60:20:20:0.05) as the mobile phase was used. The flow rate was 0.8 mL x min(-1) and UV detection was set at 274 nm.The assay displayed good linearity over the concentration ranges of 0.06-1.0 microg (r = 0.999 9, gallic acid) and 0.16-2.58 microg (r = 0.999 9, paeonol) respectively. The average recoveries (n = 9) of gallic acid and paeonol were 98.6% (RSD = 3.0%), 98.2% (RSD = 2.5%), respectively. The samples were extracted with methanol for 24 h bu maceration.The method is simple, accurate and can be used for the quality study of bark of P. suffruticosa.

Methods of separating and classifying the relative amounts of two or more classes of hydrocarbon molecules in a sample are disclosed wherein an input stream comprising a mobile phase and the sample is injected into a chromatographic column, and an effluent stream exiting a chromatographic column is split into a first effluent stream and a second effluent stream. The mass of carbon in each of the classes of hydrocarbons in the first effluent stream is determined, preferably using a flame ionization detector, and the second effluent stream is directed through a variable restrictor, allowing the pressure and mass flow rate of the input stream to be independently controlled and improved results obtained. In a preferred embodiment the present invention also includes identifying the hydrocarbon molecules in the second effluent stream in order to verify the analysis or provide additional data, preferably by using an ultraviolet detector. The present invention is most preferably used to analyze the aromatic hydrocarbons in a sample of diesel fuel.

Several high performance liquid chromatographic methods for the analysis of gossypol in different kinds of sample were developed. (1) Pure gossypol: separation on C18 column with MeOH/H2O/CHCl3 (70:30:40) containing 0.1% H3PO4 as mobile phase or on SO3H column with MeOH/citrate buffer (pH 6.3) (55:45) as mobile phase was recommended. With these systems, minute amounts of contaminants difficult to separate by other HPLC systems could be determined. (2) Plant material: acetone was selected as the extraction solvent. After evaporation of acetone from the extract, the residue was redissolved in 1% HOAc in CHCl3. An aliquot of this solution was chromatographed and quantified by peak area method. The mean recovery of pure gossypol added to plant material was 91.1 +/- 1.1% (S.D.). (3) Plasma sample: a HPLC method with electrochemical detector was developed. A plasma sample with glutathione as protective agent and gossypol dimethyl ether as internal standard was introduced on to a C18 pre-column. By using column-switching technique, a certain part of the eluate containing gossypol and gossypol dimethyl ether was subjected to a C8 analytical column for further separation. MeOH/citrate buffer (pH 3.2) (80:20) was used as the mobile phase. The optimum potential for detection was +0.6 V vs. Ag-AgCl. The assay sensitivity was 5 ng/ml. This method is sensitive and selective, suitable for clinical pharmacokinetic studies.

A sensitive and selective high-performance liquid chromatographic method with electrochemical detection for the determination of gossypol in human plasma is described. Glutathione is used as a protective agent and gossypol dimethyl ether as an internal standard. Acetonitrile-treated protein-free plasma sample is first introduced on to a C18 pre-column for enrichment and clean-up. By using a column-switching technique, gossypol and the internal standard are subjected to further separation on a C8 analytical column, while the major interfering components are eliminated before entering the column. Methanol-0.1 M citrate buffer (pH 3.2) (80:20) is used as the mobile phase. The detector potential on the glassy carbon electrode is maintained at +0.6 V vs. an Ag-AgCl reference electrode. The linearity with human plasma ranged from 5 to 250 ng/ml. The absolute recoveries of gossypol and gossypol dimethyl ether were 91.3 and 97.5%, respectively, with a within-day precision of 2.5% and a day-to-day precision of 3.8%. The limit of detection is 5 ng/ml (signal-to-noise ratio = 3:1). The method is considered to be suitable for the clinical pharmacokinetic studies of gossypol.

Pharmacokinetic parameters of (+ or -)- (+)- and (-)-gossypol were determined in humans and dogs after a single oral or intravenous dose. Mean (+or- SD) oral bioavailability of (+or-)- gossypol in dogs was 30.9% +or- 16.2%. Studies in dogs who received single intravenous injections revealed that the elimination t 1/2 and volume of distribution of (=)-gossypol were 5 and 6 times those of (-)-gossypol respectively whereas total body clearance and the AUC of the 2 anantiomers were similar. Data from men receiving the compounds orally show that the average peak plasma concentration and the AUC of (+)- gossypol are significantly greater than those of the (-)-isomer. The rate constants of alpha beta ka k21 and k10 for (-)-gossypol are significantly greater than those for (+)-gossypol indicating higher rates of mass transfer of the (-)-species. In humans the elimination of t 1/2 of (+)-gossypol was 29 times as that of (-)-gossypol a difference that is more striking than that found in dogs. The elimination of t 1/2 of (+or-)-gossypol in humans averages 286 +or- 179 hours. (authors)