Separation and quantitation of long-chain free fatty acids in human serum by high-performance liquid chromatography

A rapid, simple and highly sensitive reversed-phase high-performance liquid chromatographic method is described for the separation and quantitation of fatty acids in human serum using a very reactive fluorescent labeling reagent, 9-anthryldiazomethane. Quantitative esterification proceeds at room temperature without heat or catalysis. Baseline separation of nineteen select fatty acids from a standard mixture was achieved on two C18-bonded silica columns connected in tandem using stepwise gradient elution of an acetonitrile-methanol-water mobile phase. The eluent was monitored by a fluorescence detector (maximum excitation wavelength, 365 nm; maximum emission wavelength, 412 nm). The procedure was applied to the analysis of both saturated and unsaturated long-chain free fatty acids (C8 to C22) extracted from human serum. Sera from fasting and non-fasting subjects were analyzed to show the applicability of this assay to biological samples. Detection limit and recovery of free fatty acids in serum were less than 10 pmol/microliter and greater than 92%, respectively.     

Separation and quantitation of jasmonic acid using HPTLC

A rapid, simple, and stringent protocol for the detection and quantitation of jasmonic acid (JA) is designed using high-performance thin-layer chromatography. Acidified culture filtrate of Lasiodiplodia theobromae is extracted with an equal volume of ethyl acetate and spotted on silica gel 60 F(254) foil using Linomat-5 spray-on applicator. Standard JA is also spotted either internally or adjacent to the sample, and the foils are developed with isopropanol-ammonia-water [10:1:1 (v/v)] as the mobile phase. A quantitative estimation of the separated JA is performed by measuring the absorbance at 295 nm in the reflective mode. The sensitivity of the method is improved by adding internal standard to obtain a detection limit of 1 microg. The limit of quantitation is found to be 80 microg with this method. The method is shown to have selectivity, accuracy, precision, and high sample throughput, making it useful for the routine analysis of JA in basic science and perfumery industries.     

Separation and characterization of trypsin and carboxypeptidase B-digested products of Met-Lys-human proinsulin

Met-Lys-human proinsulin could be converted into insulin in vitro with the treatment of trypsin and carboxypeptidase B (CPB). Under less effective conditions, the enzymatic reaction does not proceed perfectly, and two main bands have been identified by native-polyacrylamide gel electrophoresis (PAGE) analysis. These two main products were thus separated and purified by DEAE-Sephadex A25 chromatography in a Tris-isopropanol system with an NaCl gradient. The isopropanol and NaCl were removed by a second DEAE-Sephadex column. Native-PAGE, mass spectrometric, and amino acid composition analyses indicate that one fraction of these two major products contains human insulin and desB30-insulin and that the other fraction is a mixture of human insulin analogs, which have one more basic amino acid than human insulin owing to the unsuitable amount of proteases, especially the lack of CPB. Furthermore, both receptor binding assay and radioimmunoassay have been utilized for the activity determination, and both fractions display almost full biological activity with porcine insulin as the standard. Present results provide further evidence for the quality control of recombinant human insulin production.     

伊曲茶碱中间体杂质的分离与鉴定

伊曲茶碱是一种新型选择性腺苷A2A受体拮抗剂,用于治疗帕金森氏病和改善帕金森氏病初期运动障碍.在伊曲茶碱中间体A1(6-氨基-1,3-二乙基-2,4-(1H,3H)-嘧啶二酮)的合成过程中,碱性条件或高温条件下会伴随生成至少2种副产物,在前期研究中我们已经对该中间体合成过程中的其中一种副产物进行过研究,确定其结构为(E...     

Separation and quantitation of fatty acids by high-performance liquid chromatography

To facilitate the determination of the fatty acid composition of tissues and the investigation of fatty acid metabolism, we developed a method for the rapid separation by high-performance liquid chromatography and quantitation (by ultraviolet light absorption) of p-bromophenyl esters of fatty acids which vary in chain length from 10 to 22 carbon atoms. The utility of the method was demonstrated by evaluating the fatty acid composition of human uterine decidua vera tissue and human endometrial stromal cells that are maintained in monolayer culture.     

Separation and quantitation of hazardous wastes from abrasive blast media.

A sample of glass bead abrasive blasting material (ABM) waste, received from Robins Air Force Base (Georgia), was examined to determine whether the waste could be rendered nonhazardous by separating paint contaminants from the ABM. The sample was analyzed with size distribution and toxicity characteristics leaching procedure. A Microtrac analyzer was used to measure the size of fine particles (-325 Tyler mesh), and scanning electron microscopy analysis was performed to identify the nature of the contaminants in the ABM waste. Tests using froth flotation, magnetic separation, desliming, and acid washing were conducted to develop a process for removing the contaminants. A pilot plant test using the developed process rendered 82.1% or the ABM waste material nonhazardous.     

Separation and quantitation of urinary trypsin inhibitors by reversed-phase high-performance liquid chromatography

Summary Human urines contain a family of trypsin inhibitors (UTIs) in small quantities, which seem to be involved in important biological processes. A procedure for separation and quantitative determination of such endogenous inhibitors in human urine has been developed. The urine sample is adjusted to pH 8.3 and percolated through a trypsin-Sepharose 4B column: the inhibitors are eluted with acid solution. The eluate (1000 l) is analysed by RP-HPLC with programmed elution and ultraviolet detection (200 nm). Three principal peaks have been evidenced: they are due to the elution of urinary trypsin inhibitors (UTIs) having apparent m.w. of ca. 6000, 72000, 18000 daltons, respectively. Characteristics of the procedure are: limited sample volume (ca. 200 ml) and recovery of the global inhibition activity (95%). For each UTI determination reproducibility and linearity ranges are reported.

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Trennung und Bestimmung von Trypsin-Inhibitoren im Harn durch Umkehrphasen-HPLC

     

Separation and quantitation of alkylphosphocholines and analogues of different liposome formulations by HPTLC

High-performance thin-layer chromatographic (HPTLC) analysis of non UV-active phospholipids in biological matrixes is a common method for separation, detection, and quantitation. Liposomes containing new alkylphosphocholines and analogues with enhanced cytostatic activity had been prepared. The liposomal formulations were designed to enable the intravenous application of the alkylphosphocholines and analogues and to reduce dose-limiting toxicities observed after oral administration. For quality control the liposomes were analyzed by HPTLC for content of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), cholesterol, alkylphosphocholines, and analogues and their related compounds (main degradation products). Due to the differences in lipophily of the compounds, different mobile phases were necessary to achieve separation. Automated Multiple Development was used to reduce the number of plates and to improve the selectivity and the capacity of the chromatographic system to separate the described alkylphosphocholines and analogues from DPPG and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in one chromatographic system.     

Separation and quantitation of human plasma acid stable trypsin inhibitors by RP-HPLC

Summary Human plasma contains acid stable trypsin inhibitors (ASTIs) which are present in small quantities and are bound tightly but reversibly to the enzyme. For the analysis of such endogenous inhibitors a procedure has been proposed analogous to that described by us for urinary trypsin inhibitors (UTIs). This chromatographic procedure allows a direct measurement of such substances and avoids the remarkable losses which, as a rule, are connected with the isolation methods so far used. Two ASTIs are selectively isolated by affinity chromatography on immobilized trypsin and separated by RP-HPLC when an acidified plasma sample (1 ml) is treated. These ASTIs have apparent m.w. of ca. 72000 and 18000 daltons, respectively. A third ASTI, having apparent m.w. of ca. 6000 daltons, is evidenced when at least 20 ml of plasma sample were processed with an unusual procedure which avoids the adsorption on the protein precipitate formed in acid treatment. For each ASTI determination, reproducibility, linearity range, recovery (95%) and detection limit are reported.

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Trennung und quantitative Bestimmung von säurebeständigen Trypsininhibitoren des menschlichen Plasmas durch RP-HPLC

     

Separation and quantitation of components in FD&C Red No. 3 using capillary electrophoresis

The use of capillary electrophoresis as a technique to separate and quantitate components of FD&C Red No. 3 (erythrosine, color index No. 45430) is described. The fluorescein isomers, 2',4',5'-triiodofluorescein (2,4,5-I3F) and 2',4',7'-triiodofluorescein (2,4,7-I3F), the most abundant by-products formed during the preparation of the dye, were selected for quantitation studies. The separation of other lower halogenated impurities was also demonstrated. Electrophoretic mobility of the compounds was achieved in a 50 mM borate, 25 mM sodium dodecyl sulfate buffer at pH 9.3. The limits of quantitation were found to be 0.15% (w/w) (2,4,5-I3F) and 0.14% (w/w) (2,4,7-I3F) (relative to the mass of FD&C Red No. 3). The method is linear from 0.08 to 20.0% (w/w) for 2,4,5-I3F and between 0.06 and 17.0% (w/w) for 2,4,7-13F. In addition, relative standard deviations of 2.03 and 5.11% were determined from precision studies in the repeat analysis of FD&C Red No. 3 for 2,4,5-I3F and 2,4,7-I3F, respectively. Overall, the CE method produced data in excellent agreement with the reference HPLC method, used considerably less solvent and sample, generated less waste and was found to be considerably more cost efficient.     

Separation,detection and quantitation of peptides by liquid chromatography and capillary electrochromatography

This review discusses different liquid chromatographic and capillary electrochromatographic approaches to the separation and quantitation of peptides using silica-based and polymeric-based columns with emphasis on liquid chromatography. Mass spectrometry detection and quantitation of peptides using labeled and label-free procedures, will also be discussed, as well as the effect of amino acids’ properties on the solubility of peptides, an important parameter that influences the selection of the mobile phase. A discussion of different column packing materials, reversed-phase, cyclodextrins, macrocyclic antibiotics, porous graphitic carbon, mixed-phases, and normal-phase will be included, as well as a short discussion of multi-dimensional approaches for the separation of complex peptide mixtures.     

Separation and quantitation of the four stereoisomers of itraconazole in pharmaceutical formulations by electrokinetic chromatography

The four stereoisomers of itraconazole were resolved for the first time by EKC using a CD as chiral selector. A study on the enantiomeric separation ability of different neutral CDs was carried out. Heptakis-2,3,6-tri-O-methyl-beta-CD was shown to provide the highest values for the enantiomeric resolution. The influence of some experimental conditions, such as pH, chiral selector concentration, and temperature, on the enantiomeric separation was also studied. The use of a 100 mM phosphate buffer (pH 2.5), 30 mM in heptakis-2,3,6-tri-O-methyl-beta-CD together with an applied voltage of 30 kV and a temperature of 20 degrees C enabled the separation of the enantiomers of itraconazole with high resolutions (Rs > 3.0). Finally, the method was validated and successfully applied to the quantitation of itraconazole in three pharmaceutical formulations.     

Separation and quantitation of plasma free fatty acids as phenacyl esters by HPLC

We have developed a rapid method for the separation of plasma free fatty acids as their phenacyl esters by high-performance liquid chromatography (HPLC) using a reversed-phase (C18) column. The derivatives of series of both saturated and unsaturated fatty acids (C12:0-C22:6) are simultaneously separated within 45 min and detected with ultraviolet at 241 nm. The limit of detection of fatty acids was approximately 0.5 nmol in 20 microL injected volume of extracts, and the coefficient of variation of the present method did not exceed 3.0%. Comparison of the results of the present HPLC method with those of gas chromatography, gave very good correlations for all fatty acids in human plasma.     

Separation and detection of selenium-containing proteins in human serum

Selenium-containing proteins or their subunits in human serum were separated and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the amount of selenium in each protein band was determined by HPLC with a fluorescence detector after derivatization with 2,4-diaminonaphthalene (DAN). This procedure provides a detection limit of 0.06 ng in a linear range of 0-1.5 ng. A protein is defined as a selenium-containing protein if its mean Se content exceeds twice the detection limit (0.12 ng) and twice the standard deviation of three replicates in sample determination. At least 4 selenium-containing bands with apparent molecular masses of 57-74, 46-56, 40-42 and 21-22 kDa could be detected from human serum collected from 4 volunteers.     

Separation and quantitation of phycobiliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection

The similar electrophoretic mobilities and sizes of several of the phycobiliproteins, which are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae, render their separation and quantitation a challenging problem. However, we have developed a suitable capillary electrophoresis (CE) method that employs a phytic acid-boric acid buffer and laser-induced fluorescence (LIF) detection with a single 594 nm He-Ne laser. This method takes advantage of the remarkably high quantum yields of these naturally fluorescent proteins, which can be attributed to their linear tetrapyrrole chromophores covalently bound to cysteinyl residues. As such, limits of detection of 1.18 x 10(-14), 5.26 x 10(-15), and 2.38 x 10(-15) mol/l were obtained for R-phycoerythrin, C-phycocyanin, and allophycocyanin proteins, respectively, with a linear dynamic range of eight orders of magnitude in each case. Unlike previously published CE-LIF methods, this work describes the separation of all three major classes of phycobiliproteins in under 5 min. Very good recoveries, ranging from 93.2 to 105.5%, were obtained for a standard mixture of the phycobiliproteins, based on seven-point calibration curves for both peak height and peak area. It is believed that this development will prove useful for the determination of phycobiliprotein content in naturally occurring cyanobacteria populations, thus providing a useful tool for understanding biological and chemical oceanographic processes.     

Separation and detection of selenium-containing proteins in human serum

Selenium-containing proteins or their subunits in human serum were separated and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the amount of selenium in each protein band was determined by HPLC with a fluorescence detector after derivatization with 2,4-diaminonaphthalene (DAN). This procedure provides a detection limit of 0.06 ng in a linear range of 0–1.5 ng. A protein is defined as a selenium-containing protein if its mean Se content exceeds twice the detection limit (0.12 ng) and twice the standard deviation of three replicates in sample determination. At least 4 selenium-containing bands with apparent molecular masses of 57–74, 46–56, 40–42 and 21–22 kDa could be detected from human serum collected from 4 volunteers.