HPLC Peak Research Articles

Degradation of benomyl, picloram, and dicamba in a conical apparatus by zero-valent iron powder

Reduction of some pesticides (benomyl, picloram, and dicamba) was studied in an aerobic batch conical pilot system to investigate the disappearance of these pesticides on contact with iron powder (20 g/l, 325-mesh). Aqueous buffered solutions of the compounds were added to the system followed by zero-valent iron powder (ZVIP), and the decline in the pesticide concentrations was monitored over time. HPLC analyses show a complete disappearance of picloram (1.20 mg/l) after 20 min of reaction. Benomyl (1.00 mg/l) and dicamba (1.25 mg/l) disappear after 25 and 40 min, respectively. The t 50 values ranged from 3 to 5.5 min, and were about slightly less than the t 1/2 values reported when the log of the relative HPLC peak area was plotted versus time, where the relative peak area was calculated by dividing the measured peak area by the initial peak area. Pathways for the degradation of the studied pesticides by ZVIP are proposed.

Characterization of an in Vivo Hormonally Regulated Phosphodiesterase 3 (PDE3) Associated with a Liver Golgi-Endosomal Fraction

The biochemical properties of an in vivo hormonally regulated low Km cAMP phosphodiesterase (PDE) activity associated with a liver Golgi-endosomal (GE) fraction have been characterized. DEAE-Sephacel chromatography of a GE fraction solubilized by a lysosomal extract resulted in the sequential elution of three peaks of activity (numbered I, II, and III), while ion-exchange HPLC resolved five peaks of activity (numbered 1, 2, 3, 4, and 5). Based on the sensitivity of the eluted activity to cGMP and selected phosphodiesterase inhibitors, two phosphodiesterase isoforms were resolved: a cGMP-stimulated and EHNA-inhibited PDE2, eluted in DEAE-Sephacel peak I and HPLC peak 2 and a cGMP-, a cilostamide-, and ICI 118233-inhibited PDE3, eluted in DEAE-Sephacel peak III and HPLC peaks 3, 4, and 5. GE fractions isolated after acute treatments with insulin, tetraiodoglucagon, and growth hormone displayed an increase in phosphodiesterase activity relative to saline-injected controls, as did GE fractions from genetically obese and hyperinsulinemic rats relative to lean littermates. In all experimental rats, an increase in PDE3 activity associated with DEAE-Sephacel peak III and HPLC peaks 4 and 5 was observed relative to control animals. Furthermore, in genetically obese Zucker rats, an increase in the sensitivity of PDE activity to cilostamide and in the amount of PDE activity immunoprecipitated by an antibody to adipose tissue PDE3 was observed relative to lean littermates. These results extend earlier studies on isolated hepatocytes and show that liver PDE3 is the main if not sole PDE isoform activated by insulin, glucagon, and growth hormone in vivo.

Application of directly coupled HPLC NMR to separation and characterization of lipoproteins from human serum.

Disorders in lipoprotein metabolism are critical in the etiology of several disease states such as coronary heart disease and atherosclerosis. Thus, there is considerable interest in the development of novel methods for the analysis of lipoprotein complexes. We report here a simple chromatographic method for the separation of high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein from intact serum or plasma. The separation was achieved using a hydroxyapatite column and elution with pH 7.4 phosphate buffer with 100-microL injections of whole plasma. Coelution of HDL with plasma proteins such as albumin occurred, and this clearly limits quantitation of that species by HPLC peak integration. We also show, for the first time, the application of directly coupled HPLC 1H NMR spectroscopy to confirm the identification of the three major lipoproteins. The full chromatographic run time was 90 min with stopped-flow 600-MHz NMR spectra of each lipoprotein being collected using 128 scans, in 7 min. The 1H NMR chemical shifts of lipid signals were identical to conventional NMR spectra of freshly prepared lipoprotein standards, confirming that the lipoproteins were not degraded by the HPLC separation and that their gross supramolecular organization was intact.

Activation of Prostacyclin Synthesis by Mechanical Stimulation in the Ciliated Protozoan Tetrahymena thermophila

We detected a HPLC peak corresponding to 6-keto-PGF1α, a stable metabolite of prostacyclin (PGI2), in [1-14C]arachidonate metabolites from the ciliated protozoan Tetrahymena thermophila. Quantitative analysis of 6-keto-PGF1α by enzyme immunoassay revealed that the synthesis and release were rapidly activated by the mechanical stimulation of a short centrifugation. The activation was suppressed significantly by a cyclooxygenase inhibitor, ibuprofen, and was independent of the extracellular Ca2+. External addition of PGI2 and its stable analogue, beraprost, caused a transient increase in the tumbling frequency of swimming. Other prostanoids, PGE2 and PGF2α, have no effect on the swimming. These results indicate that a free-living ciliate, T. thermophila, synthesizes and has a specific sensitivity to PGI2.

Liquid Chromatographic Assay for a Glucose Tetrasaccharide, a Putative Biomarker for the Diagnosis of Pompe Disease

A HPLC method associated with butyl-p-aminobenzoate derivatization has been developed for the analysis of a tetraglucose oligomer, Glcα1-6Glcα1-4Glcα1-4Glc, designated Glc4, in biological fluids. This tetraglucose, normally excreted in the urine, has previously been shown to be elevated in a number of pathological conditions including Pompe disease (glycogen storage disease type II), which is caused by a deficiency of the lysosomal enzyme acid α-glucosidase. Concentrations of Glc4 in both urine and plasma were established for the age ranges of <1, 1–5, 6–10, 11–20, and >20 years, both in normal individuals and in a cohort of 21 patients with enzymatically confirmed Pompe disease. The Glc4 concentration decreased with age in both groups, but all the patients had elevated Glc4 levels compared with age-matched controls. Electrospray tandem mass spectrometry was employed to establish the homogeneity of the HPLC peak for Glc4 and to investigate the identity of other unusual oligosaccharides excreted in patient urine. Our results demonstrate that this method is suitable for application in clinical laboratories to help establish the diagnosis of Pompe disease.

The Importance Of LC-MS And LC-NMR In The Discovery Of New Lead Compounds From Plants

Rapid detection of biologically active natural products plays a key role in the phytochemical investigation of crude plant extracts. In order to perform an efficient screening of the extracts, both biological assays and HPLC analysis with various detection methods are used. Techniques such as HPLC coupled to UV photodiode array detection (LC/DAD-UV) and to mass spectrometry (LC/MS or LC/MS/MS) provide numerous on-line structural data on the metabolites prior to isolation. The recent introduction of HPLC coupled to nuclear magnetic resonance (LC/NMR) represents a powerful complement to LC/UV/MS screening. Various plant species belonging to the Gentianaceae and Leguminosae have been analysed by LC/UV, LC/MS, LC/MS/MS and LC/NMR. These hyphenated techniques allow a rapid structural determination of known plant constituents with only a minute amount of plant material. Simple bioautographic assays such as those used for screening antifungal constituents can also be performed on-line directly by collecting HPLC peaks and measuring the activity against the fungi of interest. These bioassays permit a rapid localisation of the bioactive natural products. With such a combined approach, the time consuming isolation of common natural products is avoided and an efficient targeted isolation of compounds presenting interesting spectroscopical or biological features is performed. Several representative applications of the use of LC/MS and LC/NMR for the dereplication and identification of antifungal constituents are presented in the present paper.

The combined use of high performance liquid chromatography and immuno‐biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins

HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound. Copyright © 2000 John Wiley & Sons, Ltd.

The combined use of high performance liquid chromatography and immuno-biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins.

HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.

Isolation and characterization of a peanut maturity-associated protein.

Using reversed-phase high-performance liquid chromatography (RP-HPLC), the peanut protein profile was shown to be related to the maturity, drying time, and drying procedure of the peanut. Differences were seen between (a) immature and mature seeds for untreated and windrow-dried peanuts, (b) untreated and windrow-dried peanuts for immature and mature seeds, and (c) windrow- and stackpole-dried peanuts. The most pronounced HPLC peak that increased in size as the peanut matured and decreased in size with longer drying times was isolated and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry to have a molecular weight of 62 500. Since maturity is related to the sensory quality of peanuts, this protein may be a marker for peanuts that will produce a higher quality flavor when roasted.

Comparative Metabolism and Toxicokinetics of 14C-ResorcinoI Bis-Diphenylphosphate (RDP) in the Rat, Mouse, and Monkey

As part of a comprehensive program to examine the safety and toxicokinetics of the flame retardant, resorcinol bis-diphenylphosphate (RDP), the metabolism and toxicokinetics of RDP was determined after exposure of rats and monkeys to 14C-RDP via intravenous, inhalation, oral, or dermal routes. The metabolism of RDP was also determined in mice. Blood, urine, and fecal samples were collected at specified times for the quantification of 14C levels. Expired air was collected from rats. Excreta samples were quantitatively extracted. Chromatographic profiles of urinary and fecal metabolites were generated for multiple animals from each group. The major metabolites were isolated and then purified by a multistep chromatographic process. Structures of urinary and fecal metabolites were determined by HPLC-mass spectrometry (MS), MS-MS, and MS-MS-MS techniques. The metabolic pattern in all three species is complex. In certain urine and feces samples, radioactivity was associated with more than 30 HPLC peaks. There was little interanimal variability in metabolic profiles. No differences between species or sexes were apparent. The parent molecule, RDP, was present in significant amounts only in the feces of animals exposed by inhalation or gavage; this appears to reflect unabsorbed ingested material. Structures of all major urinary and fecal metabolites (representing 5% of the administered dose) in rats and primates were identified. The major fecal metabolites were resorcinol diphenylphosphate (RDP half ester), hydroxy-RDP half ester, dihydroxy-RDP, and hydroxy-RDP. Major urinary metabolites were identified as resorcinol, resorcinyl glucuronide, and resorcinyl sulfate. A small amount of 14CO2 was expired. This study confirms that RDP is metabolized in an identical manner by rats, mice, and primates, and that the rat and mouse are appropriate surrogates (animal models) in which to assess the toxicity of RDP. In both rats and primates, the highest peak plasma concentration (Cmax) and greatest area under the curve (AUC) was obtained with intravenous (TV) administration. In rats the Cmax for inhalation, oral, and dermal exposures were 42%, 10%, and 1%, respectfully. The AUC values for these three routes were 60%, 58%, and 15% of the IV AUC. Approximately 20% of the dermal dose was absorbed in the rat whereas primates absorbed only 10% of the applied dermal dose. Tissue accumulation and retention of radioactivity was minimal, indicating complete clearance of the administered dose.

Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry.

A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.

Multiple Antimicrobial Activities of the Marine Alga Spyridia filamentosa (Ceramiaceae, Rhodophyta)

Forty eight HPLC peaks from three distinct TLC zones obtained from crude lipid extracts of the tropical marine red alga Spyridia filamentosa were assayed against five pathogenic microorganisms. Of these, nineteen showed detectable activities (seven HPLC peaks from TLC zone 5; five peaks from TLC zone 9; seven peaks from TLC zone 12). Activities by single HPLC peaks were generally against one or two assay microorganisms. The total number of biologically active compounds in a given alga is shown to be substantially greater than can be ascertained by assaying whole-algal extracts.

The combined use of high performance liquid chromatography and immuno-biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins

HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.

Capillary electrophoretic and liquid chromatographic determination of synthetic dyes in non-alcoholic drinks and wine samples

Synthetic dyes in wine, juices, lemonades, instant and non-alcoholic drinks were determined by capillary electrophoresis (CE) and liquid chromatography (HPLC) with UV-VIS diode-array detection (DAD). The results were in the very good agreement (the relative deviations D &amp;lt; 16%, D &amp;lt; 14% and D &amp;lt; 15% between CE/UV-VIS, CE/HPLC and HPLC/UV-VIS, respectively, D &amp;lt; 6% in majority of samples) with those obtained by UV-VIS spectrophotometry. A borate/phosphate buffer of pH 9.0 (12.5mM borate and 12.5mM phosphate) containing 40mM sodium dodecylsulfate (SDS) was selected as an electrolyte for CE. A 50mM phosphate buffer and 5mM tetrabutylammonium hydroxide (TBAOH) water solution of pH 4.2 in 38% (v/v) acetonitrile were used as a mobile phase for isocratic HPLC separation on Sepharon SGX C18 (3 × 150 mm, 5 µm) column. RSDs for individual colorants were &amp;lt; 0.5% for migration times, &amp;lt; 3% for electrophoretic peak areas, &amp;lt; 0.9% for retention times and &amp;lt; 2.4% for HPLC peak areas. Limits of quantitations (LOQs for 10 S/N) were 1.5–3.3 mg/l for CE when using 50 µm I.D. separation capillary or 0.2 to 0.4 mg/l for HPLC.

Understanding of peak deterioration in hyphenated speciation systems due to gas–liquid separation in the hydride generation interface

In hyphenated speciation systems with a hydride generation interface one of the processes influencing peak deterioration is gas–liquid separation. A mathematical model was developed to calculate attenuation, signal tailing and resolution loss of HPLC peaks due to gas–liquid separation. It was shown experimentally — using an HPLC–hydride generation–atomic fluorescence spectrometry system for arsenic speciation — that the mathematical model predicts peak deterioration well. This allowed us to study the parameters influencing the deterioration, viz. gas–liquid separation parameters (gas–liquid separator head space volume and purge gas volume flow-rate) and HPLC peak parameters [width (ratio) and resolution] theoretically, simulating HPLC peaks with gaussian functions.

Increased biological potency of hexafluorinated analogs of 1,25-dihydroxyvitamin D 3 on bovine parathyroid cells

1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) is known to be involved in regulating the proliferation of parathyroid cells and PTH synthesis through reactions involving its nuclear receptor. We evaluated the effects of 1,25-(OH) 2D 3 and its hexafluorinated analog, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D 3 (26,27-F 6-1,25-(OH) 2D 3), on parathyroid cells. The 1,25-(OH) 2D 3 and 26,27-F 6-1,25-(OH) 2D 3 each inhibited [ 3H]thymidine incorporation and ornithine decarboxylase (ODC) activity, which is important in cell proliferation, in primary cultured bovine parathyroid cells. The inhibitory effect of 26,27-F 6-1,25-(OH) 2D 3 on PTH secretion from parathyroid cells was significantly more potent than that of 1,25-(OH) 2D 3 between 10 −11 M and 10 −8 M. Study of 26,27-F 6-1,25-(OH) 2D 3 metabolism in parathyroid cells in vitro elucidated its slower degradation than that of 1,25-(OH) 2D 3. After 48 h of incubation with [1 β- 3H]26,27-F 6-1,25-(OH) 2D 3, two HPLC peaks, one for [1 β- 3H]26,27-F 6-1,25-(OH) 2D 3, and a second larger peak for [1 β- 3H]26,27-F 6-1,23(S),25-(OH) 3D 3, were detected. No metabolites were detected after the same period of incubation with 1,25-(OH) 2[26,27- 3H]D 3. We observed that 26,27-F 6-1,23(S),25-(OH) 3D 3 was as potent as 1,25-(OH) 2D 3 in inhibiting the proliferation of parathyroid cells. Data suggest that the greater biological activity of 26,27-F 6-1,25-(OH) 2D 3 is explained by its slower metabolisms and by the retention of the biological potency of 26,27-F 6-1,25-(OH) 2D 3 even after 23(S)-hydroxylation.

Maternal impact on egg development in Lymnaea stagnalis: a growth factor is produced by the albumen gland in the reproductive tract

Summary In the freshwater snail Lymnaea stagnalis, perivitellin fluid is the main source of nutrition for developing embryos; it contains predominantly galactogen and proteins. The fluid is produced by the albumen gland, a large exocrine organ in the female reproductive tract. To further define the protein products of this gland and provide more information about the maternal contribution to egg development, albumen glands were extracted and the extracts purified by reversed-phase HPLC. One major HPLC peak fraction exhibited neurotrophic activity when bioassayed on identified Lymnaea neurons in vitro; it has been partially sequenced and has the highest degree of sequence identity with epidermal growth factor (EGF). These results demonstrate that a growth factor with neurotrophic activity is produced by the albumen gland, and is packaged along with the eggs to serve a growth-promoting function in the embryo.

Real time quantitation of a chemical reaction by fiber optic near-infrared spectroscopy

In this paper, a quantitative method for following the progression of an industrially significant reaction using chemometrics, fiber-optic near-infrared (NIR) spectroscopy, and a single fiber transmission probe is reported. This method is based on a calibration for the reaction developed by sampling aliquots of a typical reaction for off-line HPLC analysis immediately after acquiring a spectrum. A comparison of HPLC peak areas to the spectra was used to develop a calibration for monitoring real-time product formation. A partial least squares (PLS) calibration model was developed from the characteristic reaction data and was applied to a 30-g-scale reaction. The model successfully predicted the formation of the product. An advantageous feature of this method is that although the product contained a specific absorbance in the NIR, by-product generation during the reaction could also be monitored by NIR. Based on the PLS model generated from both the product and by-product, the product concentration can be predicted directly from the spectral data with a correlation greater than 0.9, and required only a single PLS factor.

Toxic cyanobacterial blooms in Lake Varese (Italy): A multidisciplinary approach

During the summer of 1997, a toxic cyanobacterial bloom was observed in Lake Varese. The cyanobacterial community is recognized morphologically as being dominated by species belonging to the genera Aphanizomenon, Oscillatoria, Anabaena, and Microcystis. HPLC analyses were performed on phytoplankton samples collected on the lake from July to September 1997. A HPLC peak that had the same retention time as saxitoxin was detected in the sample collected on August 19. In addition, extracts from shellfish and fish tissues showed the same peak. In electrophysiological experiments, extracts of cyanobacteria showed an almost complete inhibition of Na+ channels, the typical biological effect of saxitoxin. The genomic region corresponding to the phycocyanin intergenic spacer of cyanobacteria from the last summer bloom has been characterized by means of a PCR amplification technique. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 127–134, 1999

Separation of human serum albumin components by RP-HPLC and CZE and their characterization by ESI-MS

Human serum albumin (HSA) is one of the most abundant human proteins and has been shown to be heterogeneous. A RP-HPLC method has been developed to separate HSA components in commercially available preparations. Separations were carried out on Aquapore RP-300, C8 columns using gradient elution with a combination of acetonitrile/water mobile phases containing 0.05% trifluoroacetic acid as ion-pairing agent. Optimum resolution was attained on narrow-bore columns using a stepwise, linear gradient that incorporated a shallow intermediate step of 0.20%/min in Mobile Phase B. Under similar elution conditions, separations carried out on standard-size columns showed the expected decrease in resolution due to increased peak widths. A comparative analysis of three commercial products highlighted qualitative and quantitative differences. Capillary zone electrophoresis was used for the analysis of collected RP-HPLC fractions. Results indicated that while the HPLC separation was incomplete, one of the major HPLC peaks was primarily composed of one of the three main components typically separated by CZE. ESI-MS was used to characterize the two major RP-HPLC fractions and also showed that the HPLC separation was incomplete. The MaxEnt transform of the HPLC peaks was consistent with components all being HSA and closely related derivatives.