High Performance Liquid Chromatogram Research Articles

Binding rather than metabolism may explain the interaction of two food-Grade Lactobacillus strains with zearalenone and its derivative (')alpha-earalenol.

The interaction between two Fusarium mycotoxins, zearalenone (ZEN) and its derivative (')alpha-zearalenol ((')alpha-ZOL), with two food-grade strains of Lactobacillus was investigated. The mycotoxins (2 microg ml(-1)) were incubated with either Lactobacillus rhamnosus strain GG or L. rhamnosus strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and (')alpha-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or (')alpha-ZOL by lyophilized L. rhamnosus GG and L. rhamnosus LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with (')alpha-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds.

Determination of contents of 10-Hydroxycamptothecin inCamptotheca acuminata by high-performance liquid chromatogram

The determination method of 10-hydroxycamptothecin inCamptotheca acuminatu fruits by high-performance liquid chromatogram (HPLC) was studied. The HPLC analysis was performed on a HIQ sil C18(4.6×250 mm) column with mobile phase of acetonitrile-water (3:7, V:V), flow rate 1 mL·min−1 and UV detective wavelength 266 nm. Extracting 10-hydroxycamptothecin by ultrasonic method from fruits ofC. acuminata to prepare samples for analysis was systematically discussed. The optimal extraction condition was carried out by 60% alcohol solution at 60°C for 50 minutes.

Variation, heredity and selection of effective ingredients inMagnolis officinalis of different provenances

Bark samples ofMagnolis officinalis were collected from a 7-year-old trial plantation with 13 provenances in Jingning County of Zhejiang Province on June 25, 2000. The contents of magnolol and honokiol ofM. officinalis were analyzed by the method of HPLC (High Performance Liquid Chromatogram). The results showed that such qualitave traits as the content of magnolol, content of honokiol, total content of key phenol, and the ratio of magnolo to honokiol differ significantly between the provenances. The provenances with a sharpened leaf tip from the western part of Hubei Province has a highest content of phenols, and that with a concave leaf tip from the Lushan Mountain has a lowest content of phenol. All these four qualitative traits were genetically controlled, with a heritability between 0.8342 and 0.9871 in terms of provenance. In addition, both longitudinal and latitudinal geographical variations could be found, with longitudinal variations being dominant. As a result, 3 superior provenances from Wufeng, Enshi and Hefeng of the western part of Hubei as well as 10 high-quality individuals were

Species-specific chemical compositions of defense secretions from Parafontaria tonominea Attems and Riukiaria semicircularis semicircularis Takakuwa(Polydesmida: Xystodesmidae).

Chemical analyses were conducted on defense secretions from two species of millipedes, Parafontaria tonominea and Riukiaria semicircularis semicircularis (Polydesmida: Xystodesmidae). Hydrogen cyanide was detected in both species using test papers impregnated with picric acid, while the foul odor of these millipedes changed to that of benzaldehyde. n-Hexane extracts of the two species indicated different profiles of gas liquid chromatograms. Benzaldehyde and mandelonitrile were detected as the common components in both species, while benzoyl cyanide and mandelonitrile benzoate were limited to P. tonominea. These results indicate a difference in the chemical composition of the defense secretion between the two species. The high performance liquid chromatograms of methanolic extracts from the two species looked similar, consisting only of mandelonitrile as the major and benzaldehyde as the minor components. On the other hand, benzoyl cyanide and mandelonitrile were not detectable in the methanolic extracts from P. tonominea despite their high solubility to methanol, indicating that, for some reason, the two cyanogenetics are not secreted during methanolic extraction.

Structure determination and by-product profile of the NK 2 receptor antagonist nepadutant, a bicyclic glycopeptide

We have synthesized and fully characterized the NK 2 receptor antagonist nepadutant and its by-products using nuclear magnetic resonance (NMR) and restrained molecular dynamics. The agent consists of an active bicyclic hexapeptide combined with a sugar residue. Analysis of the high-performance liquid chromatogram and the mass spectroscopy spectra yields traces of three by-products with the same molecular weight as the main product. The conformation of the molecules in the bicyclic hexapeptide segment, the active region, is well defined, whereas the sugar moiety is disordered. For the peptide region of nepadutant and all of its by-products, the NMR observables can be described by a single backbone conformation, more specifically a βI, βII-turn arrangement. The active dipeptide unit Trp–Phe occupies the i+1 and i+2 position of a βI-turn. The by-product profile is characterized by different forms of sugars which are caused mainly by isomerization in the process of ring opening.

Identification and quantitative levels of antibacterial components of some New Zealand honeys

High performance liquid chromatograms of the phenolic fraction of 19 samples of New Zealand manuka honey, some with high levels of non-peroxide antibacterial activity and some with no such activity, were identical, which indicated that phenolic components of this honey are not responsible for the presence or absence of this activity in manuka honey. Similarly, the result showed that geography does not influence the phenolic composition of manuka honey. Antibacterial bee peptides and the antibacterial β-triketone leptospermone were not detected in manuka honey. Methyl syringate constituted approximately 70% w/w of the phenolic fraction of manuka honey and can be regarded as a floral marker for this honey. High performance liquid chromatographic profiles of the phenolic components of manuka, heather, clover and beech honeydew honeys were significantly different and could be used to differentiate honeys if they can be shown to be as consistent as those of manuka honey.

High-performance liquid chromatography of non-polar retinoid isomers

Normal-phase high-performance liquid chromatograms of retinal, retinol and retinyl palmitate isomers using n-heptane– tert.-butyl methyl ether as mobile phase are presented. Methods for the synthesis of various isomers of these retinoids are described. This enables one to produce standard chromatograms and to select various isomers for cochromatography and the identification of the various peaks under study. Assignment of the peaks is based on chromatograms published previously in this journal. For the main isomers it is possible to get baseline separation of the commonly occurring isomeric forms in a reasonably short analysis time.

Hb Tsukumi [β117(G19)His→Tyr]: A New Hemoglobin Variant Found in a Japanese Male

We accidentally observed an abnormal elution pattern on high performance liquid chromatogram when we examined the Hb A1c level in a 65-year-old male patient who suffered from pneumoconiosis and alcoholic liver injury. The value of the glycated fraction was within the normal range but the elution patterns on high performance liquid chromatography varied with the glyco-hemoglobin analyzers. Isoelectrofocusing and urea-cellulose column chromatography showed an anomalous fast-moving β chain estimated at approximately 47%. The instability test of the hemolysate was slightly positive. Structural analysis demonstrated that the mutant was consisted by a substitution of His→Tyr at β117. This new variant was named Hb Tsukumi for the place of residence of the patient. Additionally, the nucleotide sequence showed a change of C→T |AC(His)→TAC (Tyr)] at the first base in the 117th codon of the β gene.

Quantitative resolution of overlapping tailing peaks obtained by diode-array detector high performance liquid chromatography in the absence of pure standards using simple chemical knowledge

The diode-array high performance liquid chromatograms of 10 samples of various mixtures of the polyaromatic hydrocarbons 1,2-benzanthracene and chrysene were recorded in different proportions ranging from 1∶1 to 1∶0.001. A new approach to deconvolution based upon determining purity curves, correct selection of points in time and wavelengths, and multiple linear regression is proposed. The key to the algorithm is to select those statistically acceptable solutions for which the recovered spectra are positive and the chromatographic peaks are unimodal. It is shown that these two constraints reduce the range of solutions significantly and in eight of the samples result also in good estimates of relative chromatographic peak area, demonstrating that simple chemical information when combined with statistical methods can result in quantitative resolution in the absence of external standards.

High level of aspartic acid-bond isomerization during the synthesis of an N-linked tau glycopeptide.

An increased degree of utilization of the potential N-glycosylation site in the fourth repeat unit of the human tau protein may be involved in the inability of tau to bind to the corresponding tubulin sequence(s) and in the subsequent development of the paired helical filaments of Alzheimer's disease. To model these processes, we synthesized the octadecapeptide spanning this region without sugar, and with the addition of an N-acetyl-glucosamine moiety. The carbohydrate-protected, glycosylated asparagine was incorporated as a building block during conventional Fmoc-solid phase peptide synthesis. While the crude non-glycosylated analog was obtained as a single peptide, two peptides with the identical, expected masses, in approximately equal amounts, were detected after the cleavage of the peracetylated glycopeptide. Surprisingly, the two glycopeptides switched positions on the reversed-phase high performance liquid chromatogram after removal of the sugar-protecting acetyl groups. Nuclear magnetic resonance spectroscopy and peptide sequencing identified the more hydrophobic deprotected peak as the target peptide, and the more hydrophilic deprotected peak as a peptide analog in which the aspartic acid-bond just preceding the glycosylated asparagine residue was isomerized resulting in the formation of a beta-peptide. The anomalous chromatographic behavior of the acetylated beta-isomer could be explained on the basis of the generation of an extended hydrophobic surface which is not present in any of the other three glycopeptide variants. Repetition of the syntheses, with altered conditions and reagents, revealed reproducibly high levels of aspartic acid-bond isomerization of the glycopeptide as well as lack of isomerization for the non-glycosylated parent analog. If similar increased aspartic acid-bond isomerization occurs in vivo, a protein modification well known to take place for both the amyloid deposits and the neurofibrillary tangles in Alzheimer's disease, this process may explain the aggregation of glycosylated tau into the paired helical filaments in the affected brains.

Substitution and Reduction of Platinum(IV) Complexes by a Nucleotide, Guanosine 5'-Monophosphate.

A series of Pt(IV) anticancer complexes with different reduction potentials has been investigated for their reactivity toward 5'-guanosine monophosphate (5'-GMP). The Pt(IV) complexes studied were Pt(IV)(trans-d,l)(1,2-(NH(2))(2)C(6)H(10))Cl(4) (tetraplatin, Pt(IV)(dach)Cl(4); dach = diaminocyclohexane), cis,trans,cis-[Pt(IV)((CH(3))(2)CHNH(2))(2)(OH)(2)Cl(2)] (iproplatin, Pt(IV)(ipa)(2)(OH)(2)Cl(2); ipa = isopropylamine), cis,trans,cis-[Pt(IV)(en)(OH)(2)Cl(2)] (Pt(IV)(en)(OH)(2)Cl(2); en = ethylenediamine), Pt(IV)(en)Cl(4), and cis,trans,cis-[Pt(IV)(en)(OCOCH(3))(2)Cl(2)] (Pt(IV)(en)(OCOCH(3))(2)Cl(2)). The reactivity was monitored by the decreased (1)H NMR peak intensity at 8.2 ppm due to H8 of free 5'-GMP and the increased intensity of a new peak around 8.6 ppm due to H8 of 5'-GMP bound to Pt(II). The reactivity followed the order of cathodic reduction potentials of the Pt(IV) complexes: Pt(IV)(dach)Cl(4) (-90 mV) >> Pt(IV)(en)Cl(4) (-160 mV) > Pt(IV)(en)(OCOCH(3))(2)Cl(2) (-546 mV) > Pt(IV)(ipa)(2)(OH)(2)Cl(2) (-730 mV). The most reactive complex, Pt(IV)(dach)Cl(4), showed an additional weak peak at 9.2 ppm due to H8 of the 5'-GMP bound to the Pt(IV) complex, indicating the existence of a Pt(IV) intermediate. (1)H NMR, UV/visible absorption spectra, and high-performance liquid chromatograms suggest that the final product is Pt(II)(dach)(5'-GMP)(ox5'-GMP), where ox5'-GMP is oxidized 5'-GMP. A plausible mechanism is that there is an initial substitution of one Pt(IV)/ligand by a 5'-GMP molecule, followed by a two-electron reduction, and finally a second substitution by another 5'-GMP. In the presence of excess 5'-GMP (at least 20-fold), ox5'-GMP seems to be replaced by 5'-GMP to form Pt(II)(dach)(5'-GMP)(2). UV/visible absorption spectroscopy shows that the formation of the Pt(IV) intermediate by substitution is a very slow process followed by reduction. The reduction is characterized by a relatively fast exponential decay. The addition of a small amount of cis-[Pt(II)(NH(3))(2)Cl(2)] shortened the slow formation time of the intermediate, implicating the occurrence of a Pt(II)-assisted substitution reaction. These reactions may lead to a better understanding of the anticancer activity of Pt(IV) complexes.

Evaluation of a high-pressure, high-temperature microwave digestion system

A high-pressure, high-temperature focused microwave digestion system was evaluated and compared with a conventional, closed-vessel digestion system. A suite of 18 test elements was determined in six Standard Reference Materials (Peach Leaves, Orchard Leaves, Oyster Tissue, Bovine Liver, Bituminous Coal, and River Sediment) by inductively coupled plasma atomic emission spectrometry and electrothermal atomic absorption spectrometry. Observation of the graphite furnace background absorbance for the digestates, and their high-performance liquid chromatograms, demonstrate that the high-pressure, high-temperature system results in a more complete destruction of the sample matrix. The high-temperature system also shows higher accuracy for the test metals in the reference materials (average error 7.1%) than the conventional system (11.8%). Finally, the high-temperature system has a per-sample analysis time (7.5 min) that is 10 min faster than the conventional system. On the negative side, the high-temperature system is not capable of monitoring temperature during a digestion program. As a result, vessel rupture is common during method development. This results in complete destruction of the vessel, and the emission of potentially dangerous exhaust fumes into the laboratory environment.

An On-Line Wavelet Transform for De-Noising of High Performance Liquid Chromatograms

ABSTRACT A novel algorithm of wavelet transform, called the on-line wavelet transform, which can be used in on-line processing of signals of an instrumental analysis, is proposed. And application of the on-line wavelet transform in denoising of high performance liquid chromatograms was investigated. Results showed that the noise in chromatograms was cleanly removed by the method, peak position after the on-line de-noising dose not change, and the linearity of calibration curves of concentration versus peak area remained and even was improved. Quantitative determination of three mixed samples were investigated with five standard samples; the recoveries were between 94.0-105.0%.

Simultaneous extraction of selected benzodiazepines and benzodiazepine-glucuronides from urine by immunoadsorption.

A rapid and selective cleanup procedure based on immunoadsorption is described for the simultaneous extraction of diazepam and its free or glucuronidated metabolites nordiazepam, temazepam, and oxazepam from urine. The method can also be used for the extraction of lorazepam and lorazepam-glucuronide. Because the samples do not have to be hydrolyzed before extraction, valuable information is preserved. With the exception of lorazepam-glucuronide, recoveries between 86 and 100% were obtained at spiking levels up to 200 ng of benzodiazepine or glucuronide per milliliter of urine. Using methanol/water (90:10, v/v) as an eluent, the immunoadsorber could be used at least 20 times. High-performance liquid chromatograms of urine samples from patients receiving low therapeutic dosages of diazepam or lorazepam are shown to demonstrate the high purity of the extracts.

Enantioselective immunoaffinity extraction for simultaneous determination of optically active bufuralol and its metabolites in human plasma by HPLC

A combined method of immunoaffinity extraction with high-performance liquid chromatography has been developed for the enantioselective determination of bufuralol and its metabolites in human plasma. The antibodies having high affinity toward the asymmetric center at the C-1 position of bufuralol and its 1′-oxidized metabolites and low affinity to their antipodes were elicited by immunization of rabbits with immunogens, (1 R)- and (1 S)-1′-oxobufuralol O-carboxymethyl oxime-bovine serum albumin conjugates, respectively. 0.5 ml Of the immunoaffinity adsorbent (7.6 mg·ml −1 for anti-(1 S)-antibody and 28.8 mg·ml −1 for anti-(1 R)-antibody) prepared by immobilization of an antibody was capable of retaining up to 1 μg of ( R)- and ( S)-bufuralol and up to 500 ng of other metabolites. The adsorbates were recovered quantitatively by elution with methanol–10 mM ammonium acetate buffer (pH 5) (95:5, v/v) without any interfering peaks on the high-performance liquid chromatogram. The proposed method was evaluated to be useful for the simultaneous determination of optically active bufuralol and its metabolite in plasma with acceptable recovery and precision.

Partial least-squares calibration of two-way diode-array high-performance liquid chromatograms: influence of calibration design, noise and peak separation

An approach for the calibration of two-way diode-array high-performance liquid chromatograms is described, involving unfolding a three-way data matrix and performing partial least-squares (PLS) calibration. The properties of loadings summed over time and wavelength are discussed. The influence of calibration design, noise levels and peak separation are investigated, using pseudosimulations, both by calculating prediction and test errors and by graphical representation of the summed loadings. The importance of using an independent test set is emphasized. Calibration design is shown to have a major effect both on the appearance of the loadings and on the PLS errors.

High-performance liquid chromatogram of steroids in serum of goats during the breeding season

Serum samples from two male and two female goats were analyzed by a high-performance liquid chromatographic method developed for the simultaneous determination of adrenal and gonadal steroid species. The 2 ml serum sample was extracted by a solid-phase column prior to the analysis. A C 18 column was used in separation. The separated species were monitored at 240 nm. The estimated serum concentration of progesterone from chromatograms indicated its increase during pregnancy and decrease following delivery. Testosterone and progesterone were not detected in sera of female and male goats, respectively. A cortisol peak was detectable in all serum samples. This method provides an approach for simultaneously monitoring the patterns of secretion of many steroids.

A New Fast-Moving Variant Causing Erythrocytosis and Mild Hemolysis: HB Gàmbara [β82(EF6)Lys→glu

Hb Gámbara is a new hemoglobin variant with abnormal β chains that has been found in three out of four members of a family of Lombardy origin (Gàmbara, Brescia, Northern Italy). The affected subjects led a normal life, but they had modest erythrocytosis and mild (compensated) hemolysis with slight splenomegaly. Their abnormal hemoglobin was about 52% of the total hemoglobin, and was shown to be stable by the isopropanol test. Whole blood P50 of the proband was 19.3 Torr, Bohr effect was decreased (-0.25), as well as the molar ratio between the 2,3diphosphoglycerate level and total hemoglobin of erythrocytes (0.68). The purified abnormal hemoglobin was characterized by an altered oxygen affinity, low n-factor, chloride, and 2,3-diphosphoglycerate effects. The Bohr effect was about 40% of the normal control. The abnormal hemoglobin moved faster than Hb A at alkaline electrophoresis, and split into two fractions, probably due to the formation of hybrid tetramers (α2βAβX). The reversed phase high performance liquid chromatogram from the tryptic digest of the aminoethylated abnormal β chain subunits indicated the presence of an extra peptide, βT-9,10, replacing the individual peptides βT-9 and βT-10. Finally, the proband's DNA, drawn from a suitable segment of the β structural gene (exon 2), revealed a nucleotide sequence carrying the heterozygous mutation AAG→GAG at codon 82. This led to a Lys→Glu substitution at position 82(EF6) of the β chain.

Novel immunoaffinity extraction for liquid chromatographic determination of major metabolites of 4-acetoxy-2-(4-methylphenyl)benzothiazole in plasma

Group extraction of the metabolites of 4-acetoxy-2-(4-methylphenyl)benzothiazole has been achieved through the use of an immunoaffinity adsorbent. The antisera elicited from an immunogen, 4-hydroxy-2-(4-formylphenyl)benzothiazole O-carboxymethyloxime-bovine serum albumin conjugate, were characterized to have a broad affinity spectrum for major metabolites oxidized at the 4-methyl group of the benzene moiety. One millilitre of the immunoaffinity adsorbent prepared by immobilization of antibodies (12.5 mg ml −1) was capable of retaining up to 4 μg of benzothiazoles. The adsorbates were recovered quantitatively by elution with 90% (v/v) methanol without any interfering peaks on the high-performance liquid chromatogram. The peak-height ratio of each metabolite to an internal standard was plotted against the concentration of the former substance; good linearity was observed in the range of 10–500 ng ml −1.

Investigation of window factor analysis and matrix regression analysis in chromatography

Window factor analysis (WFA), a self-modeling chemometric method for obtaining the concentration profiles of individual components from evolutionary processes, is applied to unresolved liquid chromatograms. WFA makes use of the fact that each component lies in a specific region along the evolutionary axis, called the ‘window’. The high-performance liquid chromatograms of various mixtures of toluene, ethylbenzene, m-xylene and naphthalene, in methanol solvent, were recorded with an ultraviolet diode-array detector. Although the quantitative results obtained by WFA did not agree exactly with the known concentrations, the results were in agreement with those obtained by rank annihilation factor analysis and matrix regression analysis. Derivation of matrix regression analysis, developed during this investigation, is presented.