Response surface methodology based on central composite design accompanied by multivariate curve resolution to model gradient hydrophilic interaction liquid chromatography: Prediction of separation for five major opium alkaloids.

Hydrophilic interaction liquid chromatography on bare silica presents some benefits for analysis and purification of ionizable basic alkaloids. This mode was used to separate five major opium alkaloids: morphine, codeine, thebaine, papaverine, and noscapine. Central composite design based on response surface methodology was applied for experimental design, modeling, and optimization in a single-step gradient method. The main effects and their interactions (initial percentage of modifier, changing range of modifier in run time, pH of buffer, and its concentration) were investigated in 30 experiments. Multivariate curve resolution-alternating least squares, by resolving overlapped curves, helped in the accurate calculation of baseline resolution factors to be modeled and optimized more accurately. Then three crucial resolution factors besides elution time were modeled in quadratic and cubic equations and optimized. In addition to the four factors, five extra logarithmic, and nonlogarithmic factors extracted from the four factors to give nine factors overall were inspected on mechanism of retention. It was shown that a linear combination consist of four independence variables successfully describes morphinans retentivity in a single-step gradient method.

Noscapine comes of age.

Noscapine is a phthalideisoquinoline alkaloid, which represents a class of plant specialized metabolites within the large and structurally diverse group of benzylisoquinoline alkaloids. Along with the narcotic analgesic morphine, noscapine is a major alkaloid in the latex of opium poppy (Papaver somniferum) that has long been used as a cough suppressant and has undergone extensive investigation as a potential anticancer drug. Cultivated opium poppy plants remain the only commercial source of noscapine. Despite its isolation from opium more than two centuries ago, the almost complete biosynthesis of noscapine has only recently been established based on an impressive combination of molecular genetics, functional genomics, and metabolic biochemistry. In this review, we provide a historical account of noscapine from its discovery through to initial investigations of its formation in opium poppy. We also describe recent breakthroughs that have led to an elucidation of the noscapine biosynthetic pathway, and we discuss the pharmacological properties that have prompted intensive evaluation of the potential pharmaceutical applications of noscapine and several semi-synthetic derivatives. Finally, we speculate on the future potential for the production of noscapine using metabolic engineering and synthetic biology in plants and microbes.

Principal opium alkaloids as possible biochemical markers for the source identification of Indian opium.

A total of 124 opium samples originating from different licit opium growing divisions of India were analyzed for their principal alkaloid (thebaine, codeine, morphine, papaverine, and narcotine) content by capillary zone electrophoresis (CZE) without derivatization or purification. Absence of papaverine in Bareilly, Tilhar, and most of the samples originating from Kota is a significant observation in relation to the source of Indian opium. Multiple discriminant analysis was applied to the quantitative principal alkaloid data to determine an optimal classifier in order to evaluate the source of Indian opium. The predictive value based on the discriminant analysis was found to be 85% in relation to the source of opium and the study also revealed that all the principal alkaloids have to be analyzed for source identification of Indian opium. Chemometrics performed with principal alkaloids analytical data was used successfully in discriminating the licit opium growing divisions of India into three major groups, viz., group I, II, and III. The methodology developed may find wide forensic application in identifying the source of licit or illicit opium originating from India, and to differentiate it from opium originating from other opium producing countries.

Determination of noscapine in plasma by liquid chromatography.

A liquid chromatographic method has been developed for the determination of noscapine in plasma. Noscapine and the internal standard, papaverine, were extracted into methylene chloride by column extraction. The separation was performed on a straight-phase liquid chromatographic system using a mobile phase of hexane--methanol--chloroform--diethylamine. A high detection selectivity was obtained by UV detection at 310 nm. The precision of the method was 3.8% (standard deviation) at a level of 89 ng/ml and 9.5% (standard deviation) at 5.9 ng/ml. The selectivity of the analytical method was evaluated by comparing analytical results after isolation of extracts of plasma samples on reversed- and straight-phase liquid chromatographic systems.

Application of capillary zone electrophoresis in the separation and determination of the principal gum opium alkaloids.

We describe the use of capillary zone electrophoresis (CZE) for the qualitative and quantitative determination of major alkaloids (i.e., thebaine, codeine, morphine, papavarine and narcotine) in gum opium involving the analysis of alkaloids without derivatization or purification. Three extractions with 2.5% w/v aqueous acetic acid quantitatively extracted major alkaloids. The separation was carried out by CZE using a 7:3 mixture of methanol and sodium acetate (100 mM, pH 3.1) at a potential of 15 kV, with UV detection at 224 nm. Spiking of pure reference alkaloid standards in the opium extract was used for peak identification. The influences of buffer composition, pH and voltage on the separation of alkaloids were studied. The detection limit of each alkaloid dissolved in methanol was found to be 850 ng/mL (morphine), 450 ng/mL (thebaine), 500 ng/mL (codeine), 550 ng/mL (papaverine), and 500 ng/mL (narcotine) at an injection pressure of 300 mbar (injection volume, 4 nL) with a signal-to-noise ratio of 3:1. The external standard method was used for the quantification of alkaloids. The calibration plot was based on linear regression analysis. The relative standard deviation (RSD) for peak area and migration time was in the range of 1.03-3.56% and 0.34-0.69%, respectively. Percentage compositions (g%) of opium alkaloids in five gum opium samples were found to be in the range of 14.45-15.95 (morphine), 2.0-3.45 (codeine), 1.32-2.73 (thebaine), 0.92-2.37 (papavarine), and 3.85-5.77 (narcotine). The method developed is suitable for the routine analysis of major gum opium alkaloids in samples of forensic importance.

Simultaneous assay for six alkaloids in opium, using high-performance liquid chromatography.

A method is described for the quantitative determination of morphine, codeine, cryptopine, thebaine, papaverine, and narcotine in opium by high-performance liquid chromatography. The alkaloids are isolated from a dilute acid extract by adsorption on an Amberlite XAD-2 resin column and eluted first with methanol and then with chloroform-methanol (3+1). After solvent removal by reduced pressure evaporation, the alkaloids are redissolved in chloroform-methanol (3+1). The sample solution, plus brucine as an internal standard, is injected onto a Corasil II column and eluted with hexane that is gradient programmed with a solution of chloroform-methanol-diethylamine (100+300+1). The absorbances of the separated alkaloids are continuously monitored at 254 nm, using a flow-through ultraviolet double-beam photometer.