Effects of ergot alkaloids on food preference and satiety in rabbits, as assessed with gene-knockout endophytes in perennial ryegrass (Lolium perenne)

Neotyphodium species are fungal endophytes best known for their protection of grass hosts and production of bioactive metabolites including ergot alkaloids. Perennial ryegrass-Neotyphodium sp. Lp1 symbiota that have altered ergot alkaloid profiles (resulting from knockouts in two different endophyte genes) were fed, along with controls, to rabbits to test the effects of ergot alkaloids on food preference and satiety. Interestingly, rabbits dramatically preferred plants that were endophyte-infected but free of ergot alkaloids over endophyte-free plants (P = 0.01). Accumulation of ergot alkaloids of the clavine class counteracted the added appeal of endophyte-infected plants. In satiety tests, consumption of ergovaline (the ultimate ergot pathway product in wild-type endophyte), but not of several other ergot alkaloids, during an initial meal had a negative effect on subsequent rabbit chow consumption (P < 0.05). The data indicate that clavines were sufficient to reduce the appeal of endophyte-infected grasses, whereas only ergovaline reduced appetite.     

Investigation of the metabolism of ergot alkaloids in cell culture by fourier transformation mass spectrometry

Ergot alkaloids are known toxic secondary metabolites of the fungus Claviceps purpurea occurring in various grains, especially rye products. The liver is responsible for converting the ergot alkaloids into metabolites; however, the toxic impact of these end products of metabolism is still unknown. The aim of this study was to analyze the metabolism of ergot alkaloids in colon and liver cell lines (HT-29, HepG2), as well as in human primary renal cells (RPTEC). It was shown that cells in vitro are able to metabolize ergot alkaloids, forming a variety of metabolic compounds. Significant differences between the used cell types could be identified, and a suitable model system was established using HT-29 cells, performing an intensive metabolism to hydroxylated metabolites. The formed substances were analyzed by coupling of high-performance liquid chromatography with fluorescence detection and Fourier transformation mass spectrometry (HPLC-FLD-FTMS) as a powerful tool to identify known and unknown metabolites.     

Worldwide occurrence of mycotoxins in foods and feeds--an update

In a review presented at the first FAO/WHO/UNEP Conference on Mycotoxins in 1977, the occurrence of aflatoxins, zearalenone, ochratoxin A, citrinin, trichothecenes, patulin, penicillic acid, and the ergot alkaloids was indicated to be significant in naturally contaminated foods and feeds. The information presented on aflatoxin contamination greatly exceeded that for all other mycotoxins combined. This study reviews the worldwide levels and occurrence of mycotoxins in various commodities since 1976. Comparatively few countries have lowered the acceptable levels for aflatoxins in susceptible commodities. However, intensified efforts are needed to establish control of aflatoxin levels in the global food supply, particularly in peanuts, tree nuts, corn, and animal feeds. Extensive deoxynivalenol (DON) contamination of grains, especially wheat, was demonstrated. Co-contamination of grains by Fusarium toxins, especially DON and nivalenol, with zearalenone to a lesser extent, was reported. However, more information on co-occurrence of Fusarium toxins in cereals should be developed. When contamination of feeds by ochratoxin A was significant, this toxin occurred in swine kidney and smoked meats in high levels. On the basis of occurrence and/or toxicity, patulin and penicillic acid contamination of foods does not appear to be of real concern. More recent developments suggest, however, that expanded monitoring studies of Alternaria toxins, moniliformin, citrinin, cyclopiazonic acid, penitrem A, and ergot alkaloids are indicated.     

Liquid chromatographic determination and stability of the Fusarium mycotoxin moniliformin in cereal grains

Moniliformin is a mycotoxin produced by Fusarium subglutinans and other Fusarium species. A rapid, liquid chromatographic method for its determination in corn and wheat is described. Samples are extracted in acetonitrile-water (95 + 5); following defatting with n-hexane, an aliquot of the extract is evaporated and cleaned up on small C18 and neutral alumina columns successively. Reverse-phase liquid chromatography (LC) is conducted on a C18 column with 10 or 15% methanol or acetonitrile in aqueous ion-pair reagent as mobile phase, with detection by ultraviolet absorption at 229 and 254 nm. Average recoveries of moniliformin (potassium salt) added to ground corn and wheat at levels of 0.05-1.0 micrograms/g were 80% (n = 20) and 85% (n = 12), respectively, and the limit of detection was ca 0.01-0.18 micrograms/g, depending on LC conditions. Analysis of 24 samples of wheat, 4 samples of rye, and 12 samples of corn showed moniliformin in only 2 corn samples (0.06 and 0.2 micrograms/g). Moniliformin was also detected in a sample of artificially damaged (slashed) corn (0.2 micrograms/g) and selected kernels of corn that were field-inoculated with F. subglutinans and F. moniliforme (50 micrograms/g and 0.5 micrograms/g, respectively). In stability studies, moniliformin (potassium salt, 1 microgram/g) in ground corn and ground wheat heated at 50, 100, and 150 degrees C for 0.5-2 h decomposed moderately, e.g., 55% remained in corn after 0.5 h at 100 degrees C.     

Liquid chromatographic determination of desfuroylceftiofur metabolite of ceftiofur as residue in cattle plasma

A liquid chromatographic (LC) method has been developed for the determination of the desfuroylceftiofur metabolite of ceftiofur as a residue in the plasma of animals. Plasma sample in 0.1M pH 8.7 phosphate buffer containing dithioerythritol is incubated under nitrogen for 15 min at 50 degrees C. The sample is centrifuged, charged to a C18 cartridge, and washed with 0.1M ammonium acetate. The desfuroylceftiofur residue on the cartridge is derivatized by adding 0.1M ammonium acetate containing iodoacetamide and letting the cartridge stand in the dark for 30 min. The cartridge is then drained and rinsed, and the desfuroylceftiofur acetamide is eluted with methanol. The mixture is evaporated to dryness, dissolved in pH 10.6 sodium hydroxide, and charged to a SAX cartridge. The derivative is eluted with 2% acetic acid, reduced in volume, and dissolved in mobile phase for liquid chromatography. The LC system includes a C8 column and guard cartridge with UV detection at 254 nm. The gradient mobile phase (flow rate 1 mL/min) is 0.01M pH 5 ammonium acetate programmed to 29% methanol-water (60 + 40) in 25 min. Recoveries were 90-100% with a sensitivity of 0.1 ppm or less. The procedure has been applied to the plasma of cattle, rats, horses, pigs, and dogs.     

Deoxynivalenol, deoxynivalenol-3-glucoside, and enniatins: the major mycotoxins found in cereal-based products on the Czech market

Fusarium toxins, Alternaria toxins, and ergot alkaloids represent common groups of mycotoxins that can be found in cereals grown under temperate climatic conditions. Because most of them are chemically and thermally stable, these toxic fungal secondary metabolites might be transferred from grains into the final products. To get information on the commensurate contamination of various cereal-based products collected from the Czech retail market in 2010, the occurrence of "traditional" mycotoxins such as groups of A and B trichothecenes and zearalenone, less routinely determined Alternaria toxins (alternariol, alternariol monomethyl ether and altenuene), ergot alkaloids (ergosine, ergocryptine, ergocristine, and ergocornine) and "emerging" mycotoxins (enniatins A, A1, B, and B1 and beauvericin) were monitored. In a total 116 samples derived from white flour and mixed flour, breakfast cereals, snacks, and flour, only trichothecenes A and B and enniatins were found. Deoxynivalenol was detected in 75% of samples with concentrations ranging from 13 to 594 μg/kg, but its masked form, deoxynivalenol-3-β-d-glucoside, has an even higher incidence of 80% of samples, and concentrations ranging between 5 and 72 μg/kg were detected. Nivalenol was found only in three samples at levels of 30 μg/kg. For enniatins, all of the samples investigated were contaminated with at least one of four target enniatins. Enniatin A was detected in 97% of samples (concentration range of 20-2532 μg/kg) followed by enniatin B with an incidence in 91% of the samples (concentration range of 13-941 μg/kg) and enniatin B1 with an incidence of 80% in the samples tested (concentration range of 8-785 μg/kg). Enniatin A1 was found only in 44% of samples at levels ranging between 8 and 851 μg/kg.     

Determination of ergotamine tartrate in tablets by liquid chromatography with fluorescence detection

A method is presented for the liquid chromatographic (LC) determination of ergotamine tartrate in tablets that is applicable even in the presence of other ingredients such as phenobarbital, belladonna alkaloids, and caffeine. The sample is transferred to a volumetric flask, a small volume of formic acid is added to dissolve and stabilize the ergotamine, and the solution is diluted to volume with methanol. The solution is mixed and filtered through paper. The LC system consists of a Rheodyne injector fitted with a 20 microL loop and a C18 reverse phase column; the mobile phase is acetonitrile-water-triethylamine (700 + 300 + 0.5). Ergotamine tartrate is determined fluorometrically at an excitation wavelength of 250 nm and an emission wavelength of 430 nm. Recovery studies were conducted at the 0.3 and 1.0 mg levels. Average recoveries were 99.6 and 100.8%, respectively; relative standard deviations (RSDs) were 1.08 and 2.21%, respectively. Some commercial preparations containing ergotamine tartrate in combination with other ingredients were also analyzed. The RSDs for 5 determinations of each of 2 ground composites were 0.09 and 0.34%.     

氮锌配施对冬小麦产量及土壤氮素转化相关酶活性的影响

  【目的】  锌(Zn)能够促进冬小麦对氮(N)素的吸收利用。研究氮锌配施对冬小麦土壤氮素形态转化及相关酶活性的影响,有助于探究氮锌配施促进冬小麦吸收利用氮的可能机制,为通过合理施肥提高冬小麦产量和品质提供理论依据。  【方法】  以‘郑麦379’为试材进行壤质潮土培养试验,设置CK (不施N和Zn)、Zn (施Zn 10 mg/kg)、N (施N 0.2 g/kg)、N+Zn (施N 0.2 g/kg+Zn 10 mg/kg) 共4个处理,分析了冬小麦产量及产量构成要素,测定4个生育期植株各部位N、Zn含量,土壤NO₃–-N和NH₄+-N含量及土壤硝酸还原酶、亚硝酸还原酶、脲酶和蛋白酶活性。  【结果】  与CK相比,Zn、N及N+Zn显著提高了冬小麦每盆穗数、穗粒数和籽粒产量,提高了不同时期小麦根、茎叶、穗和籽粒中N、Zn含量,且N+Zn处理的提高幅度明显高于Zn和N处理。随着冬小麦生育期的延长,各处理下土壤NO₃–-N和NH₄+-N含量有所降低,亚硝酸还原酶和脲酶活性有所提高,蛋白酶活性有所降低。N和N+Zn处理能显著提高土壤NO₃–-N含量,且N+Zn在冬小麦生育后期提高土壤NO₃–-N含量的幅度显著高于N处理。Zn、N及N+Zn处理能显著提高冬小麦生育后期土壤NH₄+-N的含量,且N+Zn处理提高的幅度高于Zn处理。Zn处理显著降低了拔节期后土壤硝酸还原酶活性,N及N+Zn处理降低了小麦生育后期土壤硝酸还原酶活性,且N+Zn降低硝酸还原酶活性的程度高于N处理;Zn、N和N+Zn处理均降低了土壤亚硝酸还原酶活性;Zn和N处理显著降低拔节期土壤脲酶的活性,但Zn、N和N+Zn处理均显著提高了土壤蛋白酶活性。  【结论】  氮锌配施提高冬小麦籽粒产量,促进冬小麦吸收土壤氮素,这是由于氮锌配施提高了土壤脲酶和蛋白酶活性,促进了土壤有机氮向铵态氮及铵态氮向硝态氮的转化,同时降低了冬小麦生育后期土壤硝酸还原酶和亚硝酸还原酶活性,抑制了硝态氮的反硝化作用,从而提高了土壤中可供冬小麦吸收的铵态氮和硝态氮含量。     

添加外源有机物和黏粒材料对沙黄土有机碳和菠菜生长的影响

快速提升贫瘠土壤的有机碳含量是改良土壤、增加土地生产力的重要途径。本研究通过温室盆栽试验，设置了沙黄土对照(CK)、沙黄土+木本泥炭(LW)、沙黄土+褐煤1(LC1)、沙黄土+褐煤2(LC2)、沙黄土+木本泥炭+红黏土黏粒(LWR)、沙黄土+木本泥炭+砒砂岩黏粒(LWS)和沙黄土+木本泥炭+膨润土(LWB)共7个处理，每个处理5次重复，研究了不同处理下菠菜生育期内(35d)生长、生理指标差异及各处理对土壤有机碳含量提升效果。结果表明：与CK相比，各处理收获期菠菜产量和土壤有机碳含量均显著增加(P<0.05),LW、LC2、LC1、LWS、LWR和LWB处理下收获期菠菜产量分别增加了18.6%、51.3%、80.8%、127.6%、148.1%和203.8%，对应处理土壤有机碳含量分别增加了92.4%、84.3%、66.8%、84.0%、116.3%和98.3%，土壤pH均有一定程度降低。与LW处理相比，补充黏粒材料后，LWS、LWR和LWB处理下收获期菠菜叶面积分别显著增加了55.0%、86.5%和98.3%(P<0.05)，各处理土壤pH、电导率、有机碳和全氮含量有一定程...     

Appraisal of Some Soil Tests for Zinc Availability to Late‐Sown Wheat Grown in Mollisols

Abstract

Five soil extractants, namely, 0.005 M diethylene triamine pentaacetic acid (DTPA) (pH 7.3), 0.005 M DTPA+1 M ammonium bicarbonate (pH 7.6), Mehlich 3, 0.01 M ethylene diamine tetraacetic acid (EDTA)+0.05 M ammonium carbonate (pH 8.6), and 1 M magnesium chloride (MgCl₂) (pH 6.0), were evaluated to predict the response of wheat to zinc (Zn) application in Mollisols. These extractants could be arranged in the following decreasing order of their Zn extracting power: Mehlich 3>0.005 M DTPA+1 M ammonium bicarbonate>0.01 M EDTA+0.05 M ammonium carbonate>0.005 M DTPA>1 M MgCl₂. The critical limits of Zn in soil, below which the yield response to late sown wheat (var. UP‐2338) to Zn application could be expected, were 0.57 mg 0.005 M DTPA (pH 7.3) extractable and 1.72 mg Mehlich 3–extractable Zn kg^?1 soil. The critical limit of Zn in whole shoot at 60 days after emergence was found to be 26.1 mg Zn kg^?1 plant tissue. The DTPA and Mehlich 3–extractable soil Zn also correlated significantly and positively with Zn concentration in whole shoot at 60 days after emergence and total Zn uptake by wheat at harvest.     

Immunochromatography of fusarochromanone mycotoxins

The present paper describes an enzyme-linked immunoassay (ELISA) used in combination with thin-layer chromatography (TLC) and liquid chromatography (LC) for determination of fusarochromanone (TDP) mycotoxins in barley, wheat, and a Fusarium culture grown in rice and corn. The mycotoxins were first extracted from the sample with 100% methanol and subjected to TLC or LC without additional cleanup treatment. Individual fractions eluted from TLC or LC were acetylated, then analyzed by ELISA. Determinations of TDP toxins at levels as low as 0.1 and 0.5 ng were achieved by ELISA in combination with LC and TLC, respectively. The detection limit for TDP-1 in barley and wheat was about 20 ppb by ELISA alone as compared with a detection limit of 5 ppb by a combination of ELISA with either TLC or LC. Overall analytical recovery (% of added) of TDP-1 added to barley and wheat at 5, 10, and 20 ppb of TDP-1 was 106.9 +/- 15.3 and 113.2 +/- 11.6 by LC-ELISA and 108.8 +/- 9.1 and 110.4 +/- 4.9 by TLC-ELISA, respectively. Analysis of extracts obtained from Fusarium equiseti R6137 grown in corn and rice by the combination of TLC and ELISA revealed that diacetyl-TDP was also produced by this fungus in addition to TDP-1 and TDP-2. Comparable results were obtained when fungal extracts were subjected to ELISA, LC, and immunochromatography (i.e., combination of ELISA with either TLC or LC).     

Liquid chromatographic determination of vitamin K1 trans- and cis-isomers in infant formula

The normal phase liquid chromatographic (LC) method for determination of trans- and cis-isomers of vitamin K1 (phylloquinone) in infant formula described here uses an Apex silica column, isocratic elution, and UV absorption detection at 254 nm. Vitamin K1 is extracted quantitatively from the product matrix by pretreating the as-fed liquid with concentrated ammonium hydroxide and methanol, and then extracting it with a 2:1 mixture of dichloromethane and isooctane. The extract is cleaned up by silica open-column chromatography and concentrated for LC analysis. For trans-vitamin K1, the method precision is less than or equal to 3.3% RSD (relative standard deviation), and the spike recovery is 98 +/- 4%. For cis-vitamin K1, the precision is less than or equal to 12% RSD, determined at levels near the detection limit, and the spike recovery is 95 +/- 9%. The detection limit is 0.3 ng for both isomers at signal/noise = 3.     

Liquid chromatographic determination of free and added niacin and niacinamide in beef and pork

A simple liquid chromatographic (LC) method is described for the determination of free and added niacin and niacinamide in meats. A sample is homogenized and extracted with water, and the water extract is centrifuged, deproteinized with zinc hydroxide, and filtered first through a fluted paper and then through a microporous filter. The filtrate is subjected to liquid chromatography with UV detection at 263 nm. Different ion-pair systems are needed for the measurement of niacin and niacinamide on a reverse phase column. Methanol-water (1 + 9) containing 5mM tetrabutyl ammonium ion is used to separate niacin. Water containing 10mM heptane sulfonic acid is recommended for niacinamide. Recoveries (CV,%) are 104.8% (2.9%) for niacin and 96.3% (2.7%) for niacinamide at a 10 mg/100 g fortification level. Detection limit is 1 mg/100 g sample for niacin and niacinamide.     

氮素形态对强筋和中筋小麦产量和品质的影响

[目的]研究不同氮素形态对强筋和中筋小麦植株生长、籽粒蛋白质含量及产量的影响,为选择适宜氮肥种类、提高氮素利用率提供科学依据.[方法]选用强筋小麦'藁优2018'和中筋小麦'济麦22'在河北邢台进行田间试验.在相同施氮量下,设置5个氮源处理:不施氮肥(CK)、酰胺态氮肥(Urea)、铵态氮肥(NH4+-N)、硝态氮肥(...     

Liquid chromatographic determination of the estrogens daidzein, formononetin, coumestrol, and equol in bovine blood plasma and urine

A liquid chromatographic (LC) method is described for the determination of the plant estrogens diadzein, formononetin, and coumestrol and the estrogenically active metabolite equol in bovine blood plasma and urine. The blood and urine samples are incubated overnight with and without beta-glucuronidase/sulfatase for analysis of both free and conjugated forms of estrogens. Samples are applied to Extrelut columns, extracted with ethyl acetate, and evaporated to dryness. Residues from urine samples are dissolved in methanol, diluted with water, acidified with HCl, and purified by injection through a Sep-Pak C18 cartridge. This eluate is used for LC analysis. Residues from blood samples are dissolved in benzene-petroleum ether (1 + 1), extracted with ammonium hydroxide, acidified with glacial acetic acid, and extracted with ethyl acetate. The ethyl acetate extract is evaporated, dissolved in 80% methanol, injected onto a LC reverse-phase column, and separated in a linear gradient system between 40 and 80% methanol in phosphate buffer. Quantitation is performed by means of UV and fluorescence responses. The method was sensitive enough to determine 0.4 ng/mL of daidzein and formononetin and 0.1 and 13 ng/mL of coumestrol and equol, respectively, in blood, and 130, 80, and 7 ng/mL of daidzein, formononetin, and coumestrol, respectively, and 4 micrograms/mL of equol in urine. The applicability of the method was checked by the determination of total and free plant estrogens in blood samples from a dairy cow fed a normal diet.     

农田表层土壤养分空间变异特性研究

为给田间养分监测设施布设方法提供依据,在陕西杨凌选取2块农田,采用12 m×12 m嵌套6 m×6 m的采样方法,采集表层土壤（0～20 cm）养分数据,运用经典统计、地统计学结合Kriging插值方法,分析农田土壤养分空间变异特征。结果表明：冬小麦抽穗期与成熟期农田表层土壤全氮（TN）变异系数<10%,为弱变异,土壤有机质（SOM）、有效磷（AP）变异系数介于10%与100%之间,为中等变异,有效钾（AK）和铵态氮（NH4+-N）变异系数>100%,为强变异,成熟期硝态氮（NO3--N）由强变异转为中等变异。土壤养分最优半方差模型为球状模型,作物不同生育阶段,土壤养分空间相关性存在一定的差异,土壤SOM、TN块金系数<25%,空间相关性强烈,以结构性因素为主导;冬小麦抽穗期速效态养分块金系数介于25%与75%之间,空间相关性中等,随机性因素主导,成熟期<25%,空间相关性增强。采样密度由6 m×6 m变为12 m×12 m时,变异程度保持不变,土壤养分空间变异系数差值在0.04%～59.48%范围内,成熟期2号样地的AK除外,块金系数差值在0.065%～34.177%范围内,2种采样间距获得的土壤养分空间变异特征基本一致,建议选用12 m×12 m网格。     

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.     

氮肥施用对砖红壤硝态氮和盐基离子淋失特征的影响

氮肥品种的合理选用对作物增产增收、 土壤酸化改良有重要的影响。本文以海南省海口市观澜湖采集的砖红壤为研究对象,采用室内土柱模拟试验,对尿素、 硝酸铵和硫酸铵3种氮肥处理下砖红壤硝态氮及盐基离子（Ca2+、 Mg2+、 K+、 Na+）淋失特征进行了研究。结果表明, 1）硝态氮累积淋溶量表现为硫酸铵硝酸铵尿素N0,且硝态氮的淋溶量与施肥量呈正相关关系,整个淋溶过程中硝态氮累积淋溶量（y kg/hm2）与施肥量（x kg/hm2）之间满足线性方程 y=3.3064x+315.74（R2=0.8848）; 2）尿素、 硝酸铵、 硫酸铵处理整个淋溶过程的盐基离子淋溶量均表现为 Ca2+Mg2+K+Na+,且盐基离子淋溶总量（kg/hm2）表现为硫酸铵（1821.12）硝酸铵（1080.27）尿素（872.24）N0（417.23）; 3）砖红壤盐基离子迁移速率表现为硫酸铵（26.28%）硝酸铵（13.37%）尿素（11.78%）,尿素、 硝酸铵和硫酸铵处理分别以线性方程 y=0.1178x+123.18（R2=0.9121）、 乘幂方程 y=15.634x0.4423（R2=0.9259）和对数方程 y=128.38e0.0007x（R2= 0.9244）的拟合度最高。     

黑土-春小麦中三种化学氮肥的去向

用＾１５Ｎ田间微区试验研究了黑土－春小麦中作基肥施用的尿素、碳 和硝酸钾三种氮肥的氮素去向。试验设在黑龙江省海伦市郊区,氮肥用量为纯Ｎ７５ｋｇ／ｈｍ＾２,施肥深度为１０ｃｍ。结果表明,硝酸钾和尿素的氮素利用率相当,分别为５８．４％和５５．９％,显著高于碳铵（４２．６％）。硝酸钾的土壤中的残留率（２８．７％）显著低于碳铵（３８．８％）和尿素（３８．２％）,氮素总损失在５．８％￣１８．６％之间,碳铵的     

Application of reverse phase ion-pair partition chromatography to drugs of forensic interest.

A method is described for determining a wide range of abused drugs by using 1 column, a single isocratic system, and a fixed wavelength (254 nm) ultraviolet detector. Paired-ion chromatography is performed on a reverse phase muBondapak C18 column. Acidic, basic, and neutral drugs, including their corresponding salts, can be determined without prior cleanup. A counter ion, 1-heptane sulfonate, is dissolved in the aqueous organic mobile phase to give a final pH of approximately 3.5. This technique is applicable to ergot alkaloids, phenylethylamines, opium alkaloids, local anesthetics, barbiturates, and other drugs of forensic interest. Five major opium alkaloids in gum opium, namely, morphine, codeine, thebaine, narcotine, and papaverine, can be separated in approximately 20 min.