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.

Gene actions for yield and its attributes and their implications in the inheritance pattern over three generations in opium poppy (Papaver somniferum L.).

The gene actions for yield and its attributes and their inheritance pattern based on five parameter model have been explored in four single crosses (NBIHT-5 × NBIHT-6, NBIHT-5 × NBMHT-1, NBMHT-1 × NBIHT-6 and NBMHT-2 × NBMHT-1) obtained using thebaine rich pure lines of opium poppy (Papaver somniferum L.) for three consecutive generations. All the traits showed nonallelic mode of interaction, however, dominance effect (h) was more pronounced for all the traits except thebaine and papaverine. The dominance × dominance (l) effects were predominant over additive × additive (i) for all traits in all the four crosses except for papaverine. The seed and opium yield, and its contributing traits inherited quantitatively. The fixable gene effects (d) and (i) were lower in magnitude than nonfixable (h) and (l) gene effects. The estimates of heterosis were also higher in comparison to the respective parents which suggested preponderance of dominance gene action for controlling most of the traits. The phenotypic coefficient of variation was marginally higher than those of genotypic coefficient of variation for all the traits. The traits thebaine, narcotine, morphine and opium yield had high heritability coupled with high genetic advance. The leaf number, branches per plant and stem diameter showed positive correlation with opium and seed yields. The selection of plants having large number of leaves, branches and capsules with bigger size would be advantageous to enhance the yield potential.

[Absorption of papaverine, laudanosine and cepharanthine across human intestine by using human Caco-2 cells monolayers model].

Absorption of papaverine (PAP), laudanosine (LAU) and cepharanthine (CEP) as some chemical constituents of traditional Chinese medicines in human intestine were studied. By using Caco-2 (the human colonic adenocarcinoma cell lines) cell monolayers as an intestinal epithelial cell model, the permeability of PAP, LAU and CEP were studied from apical side (AP side) to basolateral side (BL side) or from BL side to AP side. The three alkaloids were measured by reversed-phase high-performance liquid chromatography coupled with UV detector. Transport parameters and apparent permeability coefficients (Papp) were then calculated and compared with those of propranolol as a control substance of high permeability and atenolol as a control substance of poor permeability. The Papp values of PAP, LAU and CEP were (3.524+/-0.223) x 10(-5), (2.821+/-0.050) x 10(-5) and (6.524+/-0.052) x 10(-5) cm s(-1) from AP side to BL side, and (5.095+/-0.508) x 10(-5), (2.646+/-0.146) x 10(-5) and (5.495+/-0.036) x 10(-5) cm s(-1) from BL side to AP side, respectively. Their Papp values were identical with those of propranolol, which is a transcellular transport marker. On the other hand, the efflux transport of PAP was 1.45 times higher than its influx transport with 0.69 rate of P(app A-->B)/P(app B-->A). But P(app A-->B)/P(app B-->A) values of LAU and CEP were 1.07 and 1.19, respectively, which suggested that the efflux transport have not been involved in their absorbed mechanism in Caco-2 cells monolayers. There is a good correlation between the Papp value and apparent distribution coefficient (Log D) at pH 7.35 for the three alkaloids. PAP, LAU and CEP can be absorbed across intestinal epithelial cells, and they are completely absorbed compounds. PAP may have been involved in efflux mechanism in Caco-2 cells monolayers model from the basolateral-to-apical direction. The O/W (oil/water) partition coefficient plays key role in their transmembrane permeation.

Extraction and determination of papaverin in pericarpium papaveris using aqueous two-phase system of poly(ethylene glycol)-(NH4)2SO4 coupled with high-performance liquid chromatography.

Based on aqueous two-phase system (ATPS) of poly(ethylene glycol) (PEG)-(NH4)2SO4, a simple pretreatment approach was developed for the extraction and determination of papaverin in pericarpium papaveris. The influence factors on phase behavior of the ATPS and partition behavior of papaverin was investigated, and partition mechanism based on the hydrophobic interaction between PEG and analyte molecules was proposed. Under the optimal conditions, the extraction efficiencies for papaverin were 93-96%, and the recoveries of the added standard were 97-106% with relative standard deviations of 1.8-2.5%. Combined with a high-performance liquid chromatography (HPLC) method, this extraction technique has been successfully applied to the determination of papaverin in pericarpium papaveris with the detection limit of 2 ng mL(-1) and the linear range of 0.10-10 microg mL(-1). Compared with the conventional liquid-liquid extraction or solid-phase extraction, this method was more environmentally benign, more cost effective and much simpler due to the direct injection of the upper phase into HPLC system.

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.

An efficient immunoaffinity chromatographic method for extraction and purification of papaverine from samples of pericarpium papaveris and food products.

A highly selective immunoaffinity column was obtained by coupling anti-papaverine polyclonal antibodies to CNBr-activated Sepharose 4B. It was found that the coupling efficiency and performance of the immunoaffinity column were greatly improved by prolonging the coupling reaction time from 3 h at 20 degrees C with shaking to incubation overnight at 4 degrees C after the 3 h shaking reaction. The pH and ionic strength were observed to be the most important factors that influence the binding of papaverine to the immunoaffinity column. Using 0.01 mol/L phosphate buffered saline (PBS, pH 8.3) and methanol-water (80:20, v/v) as the loading and eluting solutions, respectively, papaverine was first retained on the column and then quantitatively eluted out with a mean recovery of 86% at a loading concentration of 1 microg/mL. When applied to real samples of pericarpium papaveris and food products, the established immunoaffinity column showed high efficiency in removing the matrix interferences in the samples and satisfactory recovery results were obtained. The method was useful for extraction and purification of papaverine from related samples.

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.