Comparison of provitamin A determination by normal-phase gravity-flow column chromatography and reversed-phase high performance liquid chromatography

Summary The provitamin A content of some food samples was determined by methods involving MgO: Hyflosupercel gravityflow column chromatography (GFCC) and reversed phase high performance liquid chromatography (HPLC), the quantitation being done by external standardization (HPLC-ES) or internal standardization (HPLC-IS) with Sudan. The results obtained with - and -carotene in carrots, -carotene and -cryptoxanthin in papaya and -carotene in tomato and kale agreed well, showing that any of the these techniques can be used, provided the analysis is done under optimum conditions. Good separation of the different provitamins using GFCC depends on the analyst's skill and visual acuity. HPLC-ES required a constant supply of provitamin standards, thus the varying purity of commercially available standards and the high instability of these compounds could pose grave problems. Due to the stability of Sudan, HPLC-IS appeared to be the method of choice although passage of the extract through a MgO: Hyflosupercel minicolumn was required prior to injection to separate chlorophylls, dihydroxy- and polyoxycarotenoids which would otherwise elute with Sudan. Nonconformity of the Sudan structure to those of the provitamins did not effect the quantitative results. The chromatographic separation, identity and quantification of the provitamins could be more easily established by using HPLC-IS, complemented with GFCC.     

A study of the behaviour of some new column materials in the chromatographic analysis ofCatharanthus alkaloids

Summary New column-packing materials specially designed for the HPLC analysis of basic compounds have been tested for the analysis ofCatharanthus alkaloids. Mobile phase optimization was performed for each column tested. The influence of mobile phase pH, nature and content of the organic modifier and salt concentration on retention, selectivity and resolution was studied. An important factor in the separation proved to be the pH of the eluent, because of the widely different pK _a values of the analytes. Complete separation was easily achieved on ODS columns, but polymeric materials also gave acceptable results. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Analysis of terpenes fromGinkgo biloba L. extracts by reversed phase high-performance liquid chromatography

Summary The main terpenes ofGinkgo biloba L. extracts (bilobalide, ginkgolide A, ginkgolide B and ginkgolide C) have been separated by isocratic elution on a 3 μm C₁₈ Spherical column using 2-propanol:water (10∶90) as eluent.     

A study of the behaviour of some new column materials in the chromatographic analysis of Cinchona alkaloids

Summary A major problem in the HPLC analysis of alkaloids is the poor peak shape and consequently low resolution, due to the interactions of the basic alkaloids with the residual acidic silanol groups of most reversed phase materials. The performance of new packing materials specially designed for the separation of basic compounds has been studied using mobile phases without the special additives commonly applied in the analysis of alkaloids. Strongly basic Cinchona alkaloids were used as test compounds. Retention characteristics and selectivities of each material were studied, after mobile phase optimisation for the column. The influence of the major factors (nature and content of the organic modifier, pH value, salt concentration) affecting resolution was studied. The mobile phases were chosen so that they could be used in thermospray LC-MS. The addition of salts to the mobile phase improves separation but in general the modification of the mobile phase gave little change in selectivity. The performance of silicabased C₁₈ material proved superior to the polymer materials tested.     

A study of the behaviour of some new column materials in the chromatographic analysis of Cinchona alkaloids

Summary A major problem in the HPLC analysis of alkaloids is the poor peak shape and consequently low resolution, due to the interactions of the basic alkaloids with the residual acidic silanol groups of most reversed phase materials. The performance of new packing materials specially designed for the separation of basic compounds has been studied using mobile phases without the special additives commonly applied in the analysis of alkaloids. Strongly basic Cinchona alkaloids were used as test compounds. Retention characteristics and selectivities of each material were studied, after mobile phase optimisation for the column. The influence of the major factors (nature and content of the organic modifier, pH value, salt concentration) affecting resolution was studied. The mobile phases were chosen so that they could be used in thermospray LC-MS. The addition of salts to the mobile phase improves separation but in general the modification of the mobile phase gave little change in selectivity. The performance of silica-based C₁₈ material proved superior to the polymer materials tested.     

高速逆流色谱与硅胶柱层析联用分离岩黄连中的2种原小檗碱型季胺生物碱

建立了分离岩黄连2种生物碱的方法。岩黄连醇提物经溶剂萃取后的水相提取物,经过硅胶柱层析粗分离,对其中一个流分采用高速逆流色谱法,以正己烷-乙酸乙酯-正丁醇-乙醇-水-氨水(体积比为0.4:1.5:4:0.4:5:0.11)为两相溶剂系统,下相为固定相,上相为流动相,通过一次高速逆流色谱,即可从64.81 mg样品中分离得到9.28 mg纯度为97.0%的去氢碎叶紫堇碱(dehydrocheilanthifoline)和4.38 mg纯度为90.7%的脱氢异阿朴卡维汀(dehydroisoapocavidine),并通过与对照品HPLC保留时间的比较,以及ESI-MS测定分子量,确认了化合物的结构。该方法降低了反复的柱层析导致的样品损失,也为上述两种化合物的药理研究提供了物质基础。     

Determination of the five major opium alkaloids by reversed-phase high-performance liquid chromatography on a base-deactivated stationary phase

Summary A reversed phase HPLC method for the separation of the five major alkaloids fromPapaver somniferum L., morphine, codeine, thebaine, papaverine and noscapine, has been developed and validated. By use of a basedeactivated silica-based stationary phase excellent peak shape was achieved for each substance. The five alkaloids were quantified by internal standardization within 20 min and with good precision. The method is applicable to opium and to poppy straw.     

Isolation and purification of phycoerythrin from red algaGracilaria verrucosa by expanded-bed-adsorption and ion-exchange chromatography

Summary R-phycoerythrin was isolated and purified fromGracilaria verrucosa on an expanded-bed adsorption column combined with ion-exchange chromatography, which can effectively solve the problem of blockage of chromatographic column due to polysaccharides during isolation and purification of phycobiliproteins. 0.1 M (NH₄)₂SO₄ proved best to elute R-phycoerythrin from the expanded-bed column, and desalted 0.1 M (NH₄)₂SO₄ eluate was used on an ion-exchange column to purify the R-phycoerythrin. Using this two-stage chromatography, the purity (OD₅₆₅/OD₂₈₀) of the R-phycoerythrin fromG. verrucosa is increased to 4.4, and the yield of purified R-phycoerythrin can reach 0.141 mg·g⁻¹ of the frozen alga.     

Retention parameters of some isomeric 2-benzoylbenzoic acids in reversed-phase ion-pair high performance thin-layer and column chromatography

Summary The 2-benzoylbenzoic acid series was investigated by reversed-phase, high-performance, thin-layer and column chromatography using various alkylammonium salts and di(2-ethylhexyl)orthophosphoric acid as polar associating reagents. The effects of the individual substituents on retention were quantified by log k and R_M values. The compounds investigated differing in molecular structure (hydrophilic and hydrophobic substituents) commonly occurring groups in drugs and biologically active substances provide information on molecular interaction in these ion-pair systems. The combined effects on retention of organic modifier and ion-pair reagent concentration were investigated.     

Investigation of Symphytum cordatum alkaloids by liquid-liquid partitioning, thin-layer chromatography and liquid chromatography-ion-trap mass spectrometry

From the alkalised crude extract of Symphytum cordatum (L.) W.K. roots, pyrrolizidine alkaloids (PAs) were extracted as free tertiary bases and polar N-oxides in a merely one-step liquid-liquid partitioning (LLP) in separation funnel and subsequently pre-fractionated by preparative multiple-development (MD) thin-layer chromatography (TLC) on silica gel plates. In this way three alkaloid fractions of different polarities and retention on silica gel plates were obtained as: the most polar N-oxides of the highest retention, the tertiary bases of medium retention, and diesterified N-oxides of the lowest retention. The former fraction was reduced into free bases by sodium hydrosulfite and purified by LLP on Extrelut-NT3 cartridge. It was further analysed together with the two other fractions by high-performance liquid chromatography (HPLC)-ion-trap mass spectrometry with atmospheric pressure chemical ionization (APCI) interface on XTerra C₁₈ column using a gradient elution. Based on MSⁿ spectra, 18 various alkaloids have been tentatively determined for the first time in this plant as the following types of structure: echimidine-N-oxide (three diasteroisomers), 7-sarracinyl-9-viridiflorylretronecine (two diasteroisomers), echimidine (two diasteroisomers), lycopsamine (two diasteroisomers), dihydroechinatine-N-oxide, dihydroheliospathuline-N-oxide, lycopsamine-N-oxide (three diasteroisomers), 7-acetyllycopsamine-N-oxide, symphytine-N-oxide (two diasteroisomers) and 2″,3″-epoxyechiumine-N-oxide.     

Non-aqueous,reversed-phase,high-performance,thin-layer chromatography and column liquid chromatography of metal complexes of porphine

Summary The chromatographic mobility of 21H, 23H-porphine and its Ni(II), Cu(II), Zn(II) and Pd(II) complexes were investigated by high-performance thin-layer chromatography on an octadecyl-bonded, silica gel plate with various polar organic solvents including alcohols, acetonitrile, dimethylsulfoxide and propylenecarbonate. The mobility generally decreases according to the central metal ion of the complex as follows: Zn(II)>(free porphine)>Ni(II)>Pd(II)>Cu(II). Methanol is a good choice of solvent for the separation of these metal porphine complexes. Successful separation of porphine and the four metal complexes is accomplished within 13 min on a LiChrosorb RP-18 column with methanol eluent.     

Optimization of throughput in preparative column luquid chromatography by column overloading and partial fractionation of the effluent

Summary The criteria for the optimization of preparative chromatography are sample throughput and product purity. It is shown for column liquid chromatography that the throughput has been maximized for a given purity by column overloading and appropriate fractionation. The size and position of the fraction must be determined by chemical analysis, since overlapping peaks under overloading conditions are usually distorted compared to the peaks of the same amounts of pure compounds. With overloading of the column larger particles can be used as column packings without reduction of the separation effect.     

Determination of caffeoylquinic acids and flavonoids inCynara scolymus L. by high performance liquid chromatography

Summary The main phenolic compounds in dried extracts fromCynara scolymus (artichoke)—monocaffeoylquinic acids, dicaffeoylquinic acid, and flavonoids–have been separated by high-performance liquid chromatography. By use of a narrow bore C₁₈ column and an acidic mobile phase this HPLC method enabled improved separation within 31 min with significantly reduced solvent consumption compared with other methods. The method was validated to demonstrate its linearity, precision, accuracy, and robustness. Twelve commercial samples were analyzed. Monocaffeoylquinic acids were the most abundant phenolic compounds; the amounts present ranged from 0.48 to 4.24%. The amounts of dicaffeoylquinic acids and flavonoids were smaller—from 0.03 to 0.52%. The method is a good combination of efficiency and economy and should be especially useful for commercial applications.     

Separation of alkylnaphthalenes by preparative liquid chromatography using column switching systems

Summary The paper describes the separation of the mixture of alkynaphthalenes from distillation cuts of a pyrolysis oil, by preparative liquid chromatography on silica. The design of the system permits the connection of the columns to form multicolumn systems.The samples were first separated on a single column. The mixtures were further separated using two-column chromatography systems.The obtained fractions were analyzed by capillary gas chromatography. In most cases a substantial increase in the concentrations of the individual components was achieved. In several cases, pure compounds have been obtained. Separation efficiency increases in the following order: single column, two directly coupled collumns, two-step switching chromatography, heartcutting.     

Coupled column liquid chromatography for the rapid determination of N-methylcarbamates and some of their main metabolites in water

Summary A sensitive and selective coupled-column liquid chromatography (LC-LC) method was developed for the trace level determination of some N-methylcarbamates and some of their main metabolites as aldicarb, aldicarb-sulphoxide, aldicarb-sulphone, carbofuran and 3-hydroxicarbofuran in drinking and ground waters. The limit of determination can be reduced to 0.1 μg.L⁻¹ by solid phase extraction with a subsequent evaporation step. Environmental samples spiked at 0.1 μg.L⁻¹ were preconcentrated off-line with graphite carbon and then analyzed by LC-LC with UV detection yielding average recoveries between 81–109% (n=5) with RSD between 5–9%. The overall procedure allowed a sample throughput of up to 30 samples per day.     

Determination of cortisol in guinea pig urine by high performance thin-layer chromatography, high performance liquid chromatography, and thin-layer chromatography/radioimmunoassay

Summary In order to collect urinary samples from unrestrained guinea pigs, animals were kept in their familiar home cages with wood shavings for bedding. Cortisol was removed from shavings by a simple washing step, and an attempt was made to measure its concentrations by high performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC), or thin layer chromatography/radioimmunoassay (TLC-RIA). After intramuscular administration of 25 mg cortisol, cortisol excretion increased from about 20–30 μg/day to 400–500 μg/day (HPTLC: 531 μg/day, HPLC: 493 μg/day; TLC-RIA: 394 μg/day). Similarly, the treatment of the animals with 20 IU ACTH resulted in an augmented cortisol excretion, with mean values of 294 μg/day (HPTLC), 256 μg/day (HPLC) and 143 μg/day (TLC-RIA), respectively. The present study shows, for the first time, that cortisol excretion in unrestrained laboratory animals can be determined. Whilst the cortisol values measured by HPTLC and HPLC agree, the amounts measured by TLC-RIA were significantly lower. These differences are probably due to the presence of substances in urine or shavings which interfere with the radioimmunological determination. Hence, cortisol should be determined either by HPTLC or HPLC. Beside having a desirable specificity, these methods are more suited than TLC/RIA for steroid analysis since they confer the possibility of measuring additional steroids (e.g. precursors and/or metabolites of cortisol) in a single urine extract. This is especially the case for the HPTLC method since substances can be transformed into fluorescent derivatives.     

Identification of the major compounds in extracts ofVerbena officinalis L. (Verbenaceae) by HPLC with post-column derivatization

Summary A qualitative reversed-phase HPLC method has been developed for the analysis of 50% MeOH extracts ofVerbena officinalis L. (Verbenaceae) leaves. The method enables separation of the main constituents: iridoids, flavonoids and phenolic acid derivatives. Simultaneous detection at different wavelengths, measurement of the UV spectrum of each separated compound during elution and co-injection of reference substances facilitated easy and rapid identification of verbenalin, hastatoside and verbascoside. As some of these compounds, mainly flavonoids, have closely related structures, however, characterization by derivatization with reagents inducing a shift of UV absorption maxima was required. This furnished additional structural information. The reagents were adapted for compatibility with the solvent system used for the chromatographic separation.     

High-throughput analytical strategy with combined planar and column liquid chromatography for improvement of the poppy (Papaver somniferum L.) with a high alkaloid content

Summary Poppy capsules with a high alkaloid content are of great importance to the pharmaceutical industry, because approximately 35 000 tons of straw (capsule with short stem), 350 tons of straw concentrate, and 1000 tons of opium are used annually for extraction of morphinane alkaloids. The combined use of four different liquid chromatographic methods is proposed for determination of alkaloid content. In the first, semi-quantitative, method screening is performed by multilayer overpressured-layer chromatography (MLOPLC) in which 142 samples are separated on silica as stationary phase within 5 s per sample. If the morphine content is >1.3% it is then measured quantitatively by NPHPTLC (normal-phase high-performance thin-layer chromatography) combined with densitometric evaluation. A second, quantitative, determination is always performed by means of a rapid RPHPLC (reversed-phase high-performance liquid chromatography) method in which the separation time is less than 4 min per sample. If the difference between the alkaloid content measured by use of the last two methods is >12% a confirmatory RPHPLC method with increased selectivity and analysis time (15 min) must be used. The high throughput strategy presented has been used for analysis of ca. 15 000 samples per year, per genotype. Systematic selection, cross-breeding, and this analytical strategy with combined planar and column liquid chromatographic methods resulted in the discovery of two new candidate cultivars with high morphine (ca. 2%) and thebaine (ca. 1.5%) content. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001.     

Determination of hypericin and pseudohypericin in extracts fromHypericum Perforatum L. and pharmaceutical preparations by liquid chromatography-fluorescence detection

Summary An isocratic reversed-phase liquid chromatographic method for the simultaneous determination of hypericin and pseudohypericin, two of the main constituents ofHypericum Perforatum L., has been developed. The compounds were eluted from an Inertsil ODS-3, column by triethylammonium acetate-methanol-acetonirile (5:15:80) eluent and detected fluorimetrically, excitation 478, emission 598 nm. Hypericin and pseudohypericin were extracted from flowring tops by Soxhlet and pseudohypericin was isolated from the extract by collecting its chromatographic peak from the eluent flow. Identification of peaks was by HPLC coupled to a diode array detector and electrospray MS. The method was applied to the determination of hypericin and pseudohypericin in plant extract and in pharmaceutical tablets. For the latter a solid-phase extraction procedure was adopted. Riboflavin (0.1 ng.μL⁻¹) was used as internal standard. The linear working range of the method is 0.025–4 ng.μL⁻¹ and limit of detection 0.2 ng injected on-column. A comparative SPE study for hypericin is presented.