Separation of carotenol fatty acid esters by high-performance liquid chromatography

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.     

Separation of pantothenic acid derivatives by reversed-phase high-performance liquid chromatography

A variant of the use of reversed-phase high-performance liquid chromatography is described which permits the separation of pantothenic acid derivatives. The stationary phase used was a μBondapak-C₁₈ (4.1 × 250 mm column; 4.6 × 50 nm precolumn). Elution was performed in the isocratic regime using as mobile phase 20 M potassium phosphate buffer (pH 5.0)-methanol (91.5:8.5). The rate of elution was 1 ml/min. Retention times in the column for phosphopantothenate, pantothenate, phosphopantetheine, CoA, and dephosphoCoA were about 3.5, 6, 10.5, 16, and 42 min, respectively. This method, with radioactive detection, can be used for the analysis of pantothenic acid derivatives in liver extracts. One hour after white rats had been injected with [¹⁴C]pantothenic acid, the abovementioned components (with the exception of dephosphoCoA) contained the label in a ratio of 4:18:54:24. Institute of Biochemistry, Academy of Sciences of the Belorussian SSR, Grodno. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 855–858, November–December, 1988.     

Separation of pantothenic acid derivatives by reversed-phase high-performance liquid chromatography

A variant of the use of reversed-phase high-performance liquid chromatography is described which permits the separation of pantothenic acid derivatives. The stationary phase used was a Bondapak-C₁₈ (4.1 × 250 mm column; 4.6 × 50 nm precolumn). Elution was performed in the isocratic regime using as mobile phase 20 M potassium phosphate buffer (pH 5.0)-methanol (91.5:8.5). The rate of elution was 1 ml/min. Retention times in the column for phosphopantothenate, pantothenate, phosphopantetheine, CoA, and dephosphoCoA were about 3.5, 6, 10.5, 16, and 42 min, respectively. This method, with radioactive detection, can be used for the analysis of pantothenic acid derivatives in liver extracts. One hour after white rats had been injected with [¹⁴C]pantothenic acid, the abovementioned components (with the exception of dephosphoCoA) contained the label in a ratio of 4:18:54:24.Institute of Biochemistry, Academy of Sciences of the Belorussian SSR, Grodno. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 855–858, November–December, 1988.     

高效液相色谱法分离2-(4-羟基苯氧基)丙酸酯的对映体

采用高效液相色谱法在手性柱上分离芳氧基苯氧基丙酸类除草剂中间体(±)-2-(4-羟基苯氧基)丙酸酯的对映体。实验结果表明,在三苯甲酸纤维素酯的手性柱上,以无水乙醇为流动相能较好地分离(±)-2-(4-羟基苯氧基)丙酸乙酯和甲酯,其分离因子α值分别为1.44和1.29;同时还发现在三苯甲酸纤维素酯的手性柱上2-取代芳氧基或芳基丙酸酯结构中的酯基团的大小对其对映体的分离有明显的影响,其中以乙基为最佳。并通过对照试验证实了2-(4-羟基苯氧基)丙酸乙酯的右旋体先流出,其左旋体后流出。     

Separation of sialyl-oligosaccharides by high-performance liquid chromatography. Application to analysis of carbohydrate units of acidic oligosaccharides obtained by hydrazinolysis of hen ovomucoid

Sialyl- oligossacharides derived from hen ovomucoid by hydrazinolysis have been separated by liquid chromatography on quaternary amine bonded silica and alkylamine modified silicas . By using a mobile phase consisting of a mixture of acetonitrile and potassium dihydrogen phosphate with 0.01% of 1,4- diaminobutane , effective resolution of high-molecular-weight monosialylated oligossacharides was achieved in less than 2 h.     

Determination of arteether in blood plasma by high-performance liquid chromatography with ultraviolet detection after hydrolysis acid

A reversed-phase high-performance liquid chromatographic method is described for determination of the antimalarial agent arteether in blood plasma based on its decomposition in acidic medium and measurement of the major decomposition product, which has been identified as an alpha,beta-unsaturated decalone. Linear calibration curves were obtained in the range 0-250 ng/ml arteether and the recovery of the drug from plasma was found to be quantitative. There is no interference from desoxyarteether, the putative major metabolite of arteether. The method has been applied to the measurement of arteether in the plasma of rats given 110 mg/kg by intramuscular injection of the drug as a solution in sunflower oil.     

Separation of keto and keto-hydroxy bile acid isomers by high-performance liquid chromatography

Since its recently alleged use in the Middle East armed conflict, the chemical agent mustard has been in the news. Exposure to mustard causes extremely painful physiological manifestations. On entering the biological system, it is metabolized and converted into various products. This paper describes a rapid screening method for the identification of some of its common metabolites.     

Separation of porphyrin isomers by high-performance liquid chromatography

A high-speed reversed-phase high-performance liquid chromatographic method using an octadecylsilyl 3 cm long (3 microns particle size) column to separate the free acids of uroporphyrins I and III and coproporphyrins I and III from each other, and from the type I isomers of several other porphyrin carboxylic acids, is described. Separation of the porphyrins was achieved in less than 8 min, and injections were possible every 12 min. The detection limits of uroporphyrin, coproporphyrin, and mesoporphyrin were 75, 45, and 35 fmol (at a signal-to-noise ratio of 2), respectively. Application of the method to the determination of urinary and liver porphyrin patterns is shown.     

Amino acid analysis by high-performance liquid chromatography with methanesulfonic acid hydrolysis and 9-fluorenylmethylchloroformate derivatization

Experiments were undertaken to verify a method for complete amino acid analysis of plant and animal tissues and waste products from a single hydrolysis and high-performance liquid chromatographic run. Using methanesulfonic acid, hydrolysis of cytochrome c at 115 degrees C for 22 h yielded recoveries equal to or higher than hydrolysis at 115 degrees C for 70 h or at 150 degrees C for 22 h. Triple evacuation of the hydrolysis tube alternated with nitrogen flush gave recovery improvements over single evacuation. Refrigerated storage of samples under vacuum for up to 4 days between hydrolysis and further analysis was not different from immediate analysis. However, recoveries of several amino acids were reduced by refrigerated storage in air. Recoveries of individual amino acids were determined by hydrolysis of biological samples with and without added cytochrome c. Although recoveries from biological samples were lower for several amino acids, precision was sufficient to allow quantitation after correction for incomplete recoveries. Derivatization with 9-fluorenylmethylchloroformate (FMOC) was chosen because derivatives are formed with both primary and secondary amino acids, derivatives are quite stable, and detection may be either UV absorbance or fluorescence. Derivative yield is sensitive to the pH of the reaction mixture. A pH of 8.0 gave reproducible derivative yield for all physiological amino acids. Solvent extraction of excess FMOC, when compared to addition of amantadine to react with excess FMOC, gave both higher recoveries and greater precision. Following derivatization, samples could be kept at 4 degrees C for at least 24 h before high-performance liquid chromatographic analysis without loss of response. Derivative yield and detector response were constant across a wide range of molar ratio of FMOC to total amino acids. Gradient elution was required to separate FMOC derivatives on a reversed-phase column. The capability of the pumping system to produce exponential gradients permitted rapid and easy fine-tuning of the gradient.     

Separation of citric acid cycle intermediates by high-performance liquid chromatography with ion pairing

A method to separate underivatized tricarboxylic acid cycle intermediates within 20 min using the commonly available C15 high-performance liquid chromatography column has been developed. Ion pairing using tetrabutylammonium cations and isocratic conditions is used to separate the intermediates which are then detected at 210 nm. Separation was optimized by altering pH, the concentration of sodium sulfate and the pairing ion. This technique permits the detection of as little as 120 nmol of citrate to 0.5 nmol of fumarate. Physiological samples of rat liver mitochondria, human urine, and orange juice were analyzed.     

Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by high-performance capillary electrophoresis and high-performance liquid chromatography

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltaHexAFrc-GalNAc for K4 and deltaHexA-GalNAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 micro g for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 micro g for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.     

Determination of methylmalonic acid after diazonium derivatization by high-performance liquid chromatography

The use of a single prederivatization step in conjunction with high-performance liquid chromatography (h.p.l.c.) is described for the determination of methylmalonic acid (MMA). The method is based on the reaction of MMA and 4-diazobenzenesulfonic acid, which produces a derivative that has a molar absorptivity of about 9 × 10³ l mol^?1 cm^?1 at 353 nm. The derivatization reaction is optimized for various parameters. A reagent concentration of 3.3 mM at a reaction pH of 4.6 and a temperature of 100°C are optimal. The reaction product is separated from the excess of reagent and other interfering components by using a polystyrene-divinylbenzene column and a highly aqueous mobile phase. After a simple clean-up step, it is possible to quantify MMA in urine at about 0.8 mg l^?1 with linear response up to 32 mg l^?1.     

Separation of pyrimidine derivatives by reversed-phase high-performance liquid chromatography

Chromatographic behavior and separation conditions of pyrimidine derivatives were studied by high-performance liquid chromatography using a reversed-phase column and a multiwave UV detector.     

Separation of cytochromes c by reversed-phase high-performance liquid chromatography

Six kinds of cytochrome c of different origin, i.e., bovine, chicken, dog, horse, rabbit and tuna, were subjected to separation by reversed-phase high-performance liquid chromatography on three commercial packing materials; octadecyl-, octyl- and cyanoalkyl-silicas. The effects of reversed-phase material, mobile phase and temperature on the separation of cytochromes c were examined. The parameters of the mobile phase were the organic modifier, the pH, the salt concentration and additives. Under optimal conditions, five of the six cytochromes c were resolved in 10 min. The relative retention values cannot be explained in terms of the relative lipophilicities of the side-chains of the amino acid residues.     

Separation of polypeptide antibiotics by reversed-phase high-performance liquid chromatography

Summary The influence of different reversed-phase packings and the addition of acidic modifiers to the mobile phase was observed on the separation of basic and neutral polypeptide antibiotics by gradient elution. A dependence of pore size, coverage, reaction type and endcapping of the packings was not observed. Nevertheless, not all reversed-phase packings were suitable for the separation of polypeptides, especially of basic molecules. The addition of phosphoric or perchloric acid to the mobile phase prevented adsorption of the basic polypeptide antibiotics on the stationary phase.