Identification of aprotinin degradation products by the use of high-performance capillary electrophoresis, high-pressure liquid chromatography and mass spectrometry.

A preparation of bovine aprotinin, bovine pancreatic trypsin inhibitor, was subjected to high-performance capillary electrophoresis (HPCE) analysis and the purity was calculated to be approximately 80%. The two dominating contaminants were integrated to approximately 7% each as compared to the intact molecule. Characterization by high-pressure liquid chromatographic (HPLC) and mass spectrometric analysis was carried out on digests of the reduced and alkylated molecules. The contaminants were identified as truncated aprotinin, missing one and two amino acids, respectively, at the C-terminus. No such structures were identified in similar amounts in preparations of recombinant aprotinin by HPLC or HPCE.     

Analysis of tea components by high-performance liquid chromatography and high-performance capillary electrophoresis

Tea is one of the most popular beverages in the world. The number of reports on the analysis of tea components, especially for catechins, has recently been increasing. We review the recent reports on the analysis of tea components using the analytical methods of high-performance liquid chromatography and high-performance capillary electrophoresis.     

Determination of flavonoids by high-performance liquid chromatography and capillary electrophoresis

The compounds of flavonoid, an important group in nature, can prevent coronary heart disease and anticancer by virtue of the characteristics of antioxidation. Nine flavonoids most often seen in grape wine, namely apigenin, baicalein, naringenin, luteolin, hesperetin, galangin, kaempferol, quercetin, and myricetine, were determined by means of high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) in this work. A successful resolution was obtained from an unusual additive of tetrahydrofuran in mobile phase by HPLC. One notable thing is that the mixture of luteolin and quercetin could be separated for the first time by HPLC. In addition, the better detection limit was still attainable even with the use of tetrahydrofuran. The detection limits of CZE performed in borate buffer were hundreds-fold better than in previous reports. Furthermore, the retention and migration behavior of the analytes studied were discussed. As the result of this study, the elution order of flavone and flavonone was reversed to the contention proposed by Wulf et al. It was predictable from the interaction with tetrahydrofuran. Consequently, the extracts from grape wine with solid-phase extraction were analyzed by developing methods of HPLC and CZE. The obtained recoveries ranged from 90 to 107% and the relative standard deviations were under 6.3%.     

Separation of stilbenes by capillary electrophoresis and high-performance liquid chromatography

Stilbenes, fluorescence whitening agents (FWAs), are usually added to cleaning agents in household and in industry. Capillary electrophoresis (CE) was often applied to separate various compounds simultaneously for its multinomial advantages. In this paper, we established analytical methods of six diaminostilbenes with CE and ion-pair chromatography (IPC). The optimum mobile phase for IPC was 11.78 mM tetrabutylammonium hydrogen sulfate (TBA) aqueous and acetonitrile. An IPC method has been developed for simple and direct separation for diaminostilbenes, anionic substances, with TBA as ion-pair reagent. Satisfactory linear ranges (7.0 x 10(-3) approximately 3.0 x 10 microg/mL), correlation coefficients (0.9992-0.9999), and detection limits (6-13 ng/mL) were obtained. Separations were also performed by capillary zone electrophoresis (CZE) using a buffer consisting of Tris (pH 10.1), n-tetradecyltrimethylammonium bromide (TTAB) and acetonitrile. A linear range of 5.0 x 10(-1) - 4.0 x 10 microg/mL, correlation coefficients between 0.9975 and 0.9998, and detection limits between 337 and 446 ng/mL were obtained. In particular, the separation of a pair of similar compounds (mass difference of 2) was achieved by addition of TTAB. The optimum analytical methods of CE and high-performance liquid chromatography (HPLC) were applied to commercial household with direct analysis and standard addition. No significant bias were shown between them by t-test at 95% confidence level.     

Analysis of carbohydrates in glycoproteins by high-performance liquid chromatography and high-performance capillary electrophoresis

We describe two methods for the analysis of oligosaccharide chains in glycoproteins by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE).O-andN-glycosidically linked oligosaccharides released from glycoproteins can be identified as their borohydride-reduced forms by anion-exchange HPLC with pulsed amperometric detection.N-Glycosidically linked oligosaccharides can also be analyzed as 2-aminopyridine derivatives by HPCE in direct zone electrophoresis mode in an acidic phosphate buffer and zone electrophoresis mode as borate complexes in an alkaline buffer. We also present a convenient procedure for the analysis of the constituent monosaccharides of these oligosaccharides chains by HPLC based on reversed-phase partition mode as 1-phenyl-3-methyl-5-pyrazolone derivatives.     

Quantitation of insulin injection by high-performance liquid chromatography and high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was evaluated as a potential technique for the regulatory analysis of commercial dosage forms of insulin. A comparison was made to a liquid chromatographic analysis presently being proposed as an official monograph in the United States Pharmacopeia. The salient points of this comparison were accuracy, precision and ease of use. Both authentic (i.e. single blind, spiked) samples and commercial pharmaceutical formulations (injections) were examined. Chromatographic analyses of both commercial formulations and authentic samples were characterized by good precision, with accuracy being supported by results from authentic (spiked) samples. Conventional HPCE (by which is meant a non-micellar electrolyte used with an uncoated, unmodified fused-silica capillary) achieved reasonable accuracy, but less than impressive precision, when applied to authentic samples. When used for commercial formulations, this type of HPCE did not produce a level of accuracy suitable for regulatory purposes, even with the use of an internal standard.     

Separation of friend erythroleukaemic cell histones and high-mobility-group proteins by reversed-phase high-performance liquid chromatography

A procedure for the rapid separation of histones and high-mobility-group (HMG) proteins from Friend erythroleukaemic cells (line F4N) by reversed-phase high-performance liquid chromatography is reported. By using a Nucleosil 300-5 C₄, column and a multistep water—acetonitrile gradient containing 0.1% trifluoroacetic acid, the HMG-1 and HMG-2 proteins, several his subtractions including H1⁰, H4, H2B, two H2A variants and two H3 subtractions were separated. Under changed conditions, by applying a varied acetonitrile gradient system, even two H2B variants were fractionated. The methods described seem to be a real alternative to the time-consuming polyacrylamide gel electrophoresis.     

Identification of triacylglycerols by high-performance liquid chromatography-gas-liquid chromatography and liquid chromatography-mass spectrometry

Triacylglycerols from rat adipose tissue were chromatographed by high-performance liquid chromatography (HPLC), with a gradient of propan-2-ol in acetonitrile as the mobile phase. Fractions of the material eluting from the column were collected and analysed by automated gas - liquid chromatography of the fatty acid methyl esters obtained after transmethylation. Triacylglycerols were identified by using a combination of their fatty acid content and elution time from the HPLC column. Fractions corresponding to whole peaks or groups of peaks were also collected and re-chromatographed on a liquid chromatography - mass spectrometry system equipped with a belt interface. For most triacylglycerols, good agreement was obtained between the two methods, although mass spectrometric identification of the early eluting peaks was complicated by poor resolution of the triacylglycerols on the HPLC system.     

Determination of linear alkylbenzenesulfonates by high-performance liquid chromatography and capillary zone electrophoresis

High-performance liquid chromatography and capillary zone electrophoresis have been used for the separation of homologues and structural isomers of linear alkylbenzenesulfonates with subsequent UV detection. The analytical advantages of these techniques are applied to the analysis of technical products containing high amounts of LAS as well as river water samples with very low LAS concentrations using preconcentration steps.Dedicated to Professor Dr. H. Kriegsmann on the occasion of his 70th birthday     

Determination of drug-protein interactions by combined microdialysis and high-performance liquid chromatography

Summary A simple and rapid method for determination of the parameters of the interaction between drugs and protein, including the association constant and the number of binding sites, has been developed by use of a microdialysis sampling technique combined with high-performance liquid chromatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazepine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as examples) were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. The microdialysis probe was the used to sample the mixed CBZ-HSA solution at a perfusion rate of 1 μL min⁻¹. The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance liquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; theRSD ofR was approximately 1.85%. The estimated association constant (K) and the number of the binding sites,n, on one molecule of HSA were 1.06×10⁴ M⁻¹ and 0.880, respectively, which is in good agreement with the literature values determined by high-performance frontal analysis. The potential use of microdialysis is also discussed.     

Identification of cosmetic dyes by ion-pair reversed-phase high-performance liquid chromatography

A method based on ion-pair reversed-phase high-performance liquid chromatography with detection at four wavelengths between 400 and 600 nm is reported for the separation and identification of the most common synthetic colour additives in cosmetic products. All the dyes generally employed in the U.S.A. and almost all those in current use in cosmetics in the European Community have been taken into account. The chromatography was performed on a C8 bonded silica packed column, with a 60-min gradient changing from 10 to 95% acetonitrile in water containing 10(-2) M sodium perchlorate (pH 3.0) as mobile phase (flow-rate 2.5 ml/min). Detection limits are in the range 20-100 ng for all dyes investigated. The method has been applied to the analysis of commercial lipsticks.     

Identification and quantification of the constituents of madder root by gas chromatography and high-performance liquid chromatography

The possibilities in the identification and quantitation of the constituents of Rubia tinctorum L.'s root, called also madder root, was described and compared by gas chromatography (GC)-MS, high-performance liquid chromatography (HPLC)-UV/photodiode array detection (DAD) and HPLC-MS: chromatographic analyses were carried out in parallel, from the same samples/extracts/hydrolyzates. Anthraquinone glycosides, anthraquinones, carboxylic acids and sugars were determined directly in the presence of the matrix and in its extracts without and subsequently to hydrolyses. Hydrolyses were performed as a function of time, with hydrochloric and trifluoroacetic acids, as well as enzymatically. Data revealed that as hydrolyzing agent trifluoroacetic acid is to be preferred. Madder root's anthraquinones (pseudopurpurin/purpurin, alizarin, lucidin, munjistin, nordamnacanthal) were identified on the basis of their absorption spectra (HPLC-DAD) and fragmentation patterns by GC-MS and HPLC-MS, equally. Reproducibility of anthraquinone's quantitation, by HPLC-DAD and GC-MS, in the concentration ranges of 4 x 10(-5) to 3 x 10(-2)g/g dried sample, provided an average reproducibility of 4.2% (varying between 0.9 and 9.4% relative standard deviation (RSD percentages)). Carboxylic acids (malic, citric, quinic, rosmarinic acids) and saccharides (xylose, ribose, fructose, glucose, sucrose, primverose) were quantified as their trimethylsilyl (oxime) ether/ester derivatives by GC-MS, in the concentration ranges of 10(-5)g to 10(-2)g/g dried sample, with an average reproducibility of 4.7% RSD.     

Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillary electrophoresis

This paper describes the simultaneous determination of catechins and theaflavins in green and black teas, using reversed-phase high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The tea polyphenols analyzed included (+)-catechin, catechin gallate, (-)-epicatechin, epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate, theaflavin, theaflavin-3-monogallate, theaflavin-3'-monogallate and theaflavin-3,3'-gallate. These polyphenols together with six other tea ingredients such as caffeine, adenine, theophylline, quercetin, gallic acid and caffeic acid were separated within 27 min by HPLC and in less than 10 min by CE. The optimal analytical conditions of both chromatographic methods were investigated for the convenience and reliability for routine analysis. Both HPLC and CE were found to be reliable and compatible. The reproducibility of the within-day assay using both methods was generally >90%. The day-to-day variation of retention time was <5% for HPLC, while the variation of migration time for CE was <2%. The analysis time of CE was three-times faster, however it is five-times less sensitive than HPLC, which has detection limits of 0.05 microg/ml and 0.5 microg/ml for catechins and theaflavins, respectively.     

Separation of amphotericin B components by combined thin-layer and high-performance liquid chromatography

Amphotericin B has been separated into five components by the use of thin-layer chromatography (TLC) with only methanol as the developing solvent. Each TLC component was then subjected to high-performance liquid chromatography. Besides the two major components found in other studies, amphotericin B was also found to contain five other significant heptaenes, one tetraene, two fluorescent polyenes (pentaene and heptaene), and at least five other significant components which had appreciable absorbance at approximately 305 nm. In addition, amphotericin B was found to exhibit as in a previous study by other workers, only a trace amount of fluorescence.     

Identification and quantitation of phenolics from green beans by high-performance liquid chromatography

Summary High-performance liquid chromatography with diode-array detection has been used for the separation and quantitation of phenolics from fresh and processed green beans. Whole beans, pods and seeds of green beans were studied separately. Chromatographic profiles from pods were more complex than those found in seeds. Flavonol glycosides were confined to quercetin and kaempferol classes and they were typical of external parts of the fruit. Flavan-3-ols monomers ((+) catechin and (−) epicatechin) as well as procyanidins structures were identified in pods and seeds. Chromatographic profiles from processed beans revealed a polyphenolic composition similar to those found in pod fresh green beans. The chromatographic method was carefully validated in regard to precision and accuracy. High reproducibility of peak area (RSD<3%) and calibration slopes (RSD<4%) was obtained. Recoveries between 94–104% revealed good accuracy for the overall method. Application to quantitative determination in a representative number of samples allowed good knowledge of the phenolic composition of green beans (Phaseolus vulgaris v.vulgaris).     

Identification of medicinal additives in animal feedingstuffs by high-performance liquid chromatography

A method is described for the detection of 25 drugs used as prophylactics or as growth promoters in commercial animal feedingstuffs. The sample was extracted with aqueous acetonitrile, the co-extractives were removed with a silica cartridge and the eluate was examined by high-performance liquid chromatography, using two columns and five mobile phases. Thirteen animal ++feedingstuffs containing different combinations of drugs were prepared and used to test the method.