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Reverse-phase chromatography of polar biological substances: separation of catechol compounds by high-performance liquid chromatography.

Catecholamines and their metabolites have been separated isocratically by reverse-phase chromatography with aqueous (no organic solvent admixed) eluents. Unlike ion-exchange or ion-pair chromatography, mixtures of both acidic and basic substances can be separated in a single chromatographic run, because the retention is governed by hydrophobic interactions between the nonpolar moiety of the solute molecules and the octadecyl-silica stationary phase. The relative retention values strongly depend on the pH of the eluent, which governs the degree of dissociation of ionogenic solutes. The reproducibility of the results and the stability and efficiency of the chromatographic systems make this approach particularly attractive for use in clinical analysis.

3,4-Dihydroxyphenylacetic Acid

Characterization of pro-opiocortin, a precursor to opioid peptides and corticotropin.

The high molecular weight (approximately 30,000) precursor to opioid activity (pro-opiocortin) previously detected in extracts of rat pituitary was digested with trypsin and the resulting peptide mixture was resolved by high-performance reverse-phase chromatography. A peak of opioid activity was eluted at the position of the nonapeptide beta-LPH (61-69), which was also the same fragment obtained by trypsin digestion of betas-lipotropin or beta-endorphin. This identified the protein as a precursor to the endorphins and Met-enkephalin. No activity was detected in the position corresponding to the Leu5 analog of betas-LPH (61-69), thus ruling out the possibility of a beta-lipotropin-like precursor to Leu-enkephalin in pituitary extracts. Pro-opiocortin and beta-lipotropin are present in rat pituitary extracts in comparable amounts, approximately 10 pmol/mg of tissue.

Adrenocorticotropic Hormone

Characterization of the 5'-terminal structure of simian virus 40 early mRNA's.

RPC-5 reverse-phase chromatography has been used to isolate fragments of simian virus 40 DNA generated by appropriate digestions with restriction endonucleases. Ten specific DNA fragments, mapping successively in a counterclock-wise direction from 0.67 to 0.515 on the simian virus 40 genome, were each hybridized to cytoplasmic mRNA obtained during the early phase of simian virus 40 infection. Primer extension methods with reverse transcriptase were used to characterize the 5' ends of two species of viral mRNA which were fractionated on sucrose gradients. Analysis of the complementary DNA products demonstrated the presence of two different spliced structures of simian virus 40 early mRNA's, both of which had the same 5'-end sequences (AUU), located at residues 18 to 20 on the viral genome. The mRNA for small-t contained a segment 588 bases in length (residues 18 to 605) spliced to residues 672. A 66-nucleotide segment rich in adenine-thymine was spliced out of this mRNA. The mRNA for large-T contained a segment 308 bases in length (residues 18 to 325) which is also spliced to residue 672. A 346-base segment was spliced from this mRNA. The results suggest that there are two levels for control of genetic expression. One would be the regulation of initiation of transcription at a common promoter; the other involves post-transcriptional splicing.

Base Sequence

A Draft Map of E. coli Proteoforms.

Top-down proteomics (TDP) enables direct characterization of intact proteoforms, providing protein-level insights into molecular diversity arising from post-translational modifications and sequence variations. Despite this advantage, proteome coverage in TDP remains limited relative to bottom-up proteomics (BUP). To expand coverage, we developed an integrated multidimensional approach combining sequential protein extraction, size-exclusion chromatography (SEC) fractionation, and capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS. This approach identified 743 proteoform families and 10,613 proteoforms from E. coli cells through hundreds of MS runs. By incorporating previous E. coli TDP data sets from our group, we identified 14,932 proteoforms from 985 proteoform families, covering 43% of the E. coli proteome. The data represent the highest proteome coverage of cells by MS-based TDP, creating a draft map of E. coli proteoforms. The results offer strong evidence that MS-based TDP can reach high proteome coverage.

Escherichia coli

The extraction and analysis of benzodiazepines in tissues by enzymic digestion and high-performance liquid chromatography.

A simple, rapid and sensitive method is described for the analysis of unchanged benzodiazepine drugs and their metabolites, in human and animal tissues, using the proteolytic enzyme subtilisin Carlsberg followed by reverse-phase liquid chromatography. The enzymic digestion yields far higher recoveries than conventional extraction methods, while the liquid chromatogrpahic analysis allows the rapid separation and detection of nanogram quantities of these drugs without the need for elaborate "clean-up" procedures.

Animals

A versatile reversed-phase liquid chromatography charged aerosol detection method for streamlined monitoring of QS-21 content and stability in liposomal adjuvant formulations.

Identifying and quantifying an active adjuvant along with its degradants in drug formulations is essential for ensuring the safety and efficacy of the drug product. QS-21 is a potent adjuvant that is being evaluated in several clinical trials and is currently formulated in licensed vaccines that protect against shingles, malaria, and RSV. In aqueous environments, QS-21 is subject to hydrolytic degradation that is influenced by pH and temperature, resulting in the formation of a degradant known as QS-21 Hydrolyzed Product, QS-21 HP, which can occur during manufacturing and/or prolonged storage. The intact QS-21 and QS-21 HP induce distinct immune response profiles, making it critical to monitor the degradation of QS-21 in vaccine adjuvant formulations. To date, there has been a paucity of reliable assays for QS-21, its isomers, and degradant QS-21 HP in liposomal adjuvant formulations available that can be transferred seamlessly in quality control (QC) environments. Herein, we introduce a simple and QC-friendly liquid chromatography coupled to a charged aerosol detector (LC-CAD) enabled by stationary phase screening combined with in silico method development optimization. The method exploits 2.7&#xa0;&#x3bc;m fused-core phenyl hexyl particles, ensuring its versatility in standard and ultra-high pressure LC systems. This approach demonstrates a high correlation between predicted retention time (RT) and experimental outcomes with overall &#x2206;RT&#xa0;<&#xa0;4%. In addition, this assay shows great linearity, precision, specificity, and accuracy to advance process development characterization of new vaccine formulations.

Liposomes

Do neuroleptics prevent the penetration of dopamine agonists into the brain?

A new, specific and highly sensitive method for the determination of apomorphine based on high performance liquid chromatography on a C18 reverse-phase column, coupled with electrochemical detection has been developed. The limit of detection of the assay is approximately 0.5 ng/sample (2 pmol). Haloperidol, cis-flupenthixol, metoclopramide and reserpine prevented the accumulation of apomorphine equally in "dopaminergic" as well as in "non-dopaminergic" brain areas. The non-neuroleptic trans-isomer of flupenthixol was without effect. Suppression of the accumulation of the dopamine agonist 6,7-ADTN (administered as the prodrug dibenzoyl-6,7-ADTN) was seen after combined treatment with haloperidol or reserpine, whereas cis- and trans-flupenthixol were without effect. The results imply that certain behavioural, biochemical and neuropharmacological studies, based on apomorphine in combination with other drugs, may need reinterpretation.

Animals

Sex-dependent upregulation in oxylipins involved in inflammation resolution in the cerebellum of Niemann-Pick disease C1 mice.

Unresolved inflammation in the cerebellum is implicated in motor and cognitive decline in Niemann-Pick disease type C (NPC), a neurodegenerative lysosomal storage disorder caused by pathogenic mutations in the Npc1 gene encoding a cholesterol transporter protein. It is unclear whether unresolved inflammation in NPC stems from impairments in lipid-mediated resolution. For this reason, free lipid mediators (i.e., oxylipins) involved in inflammation resolution, as well as esterified lipid mediators known to regulate the bioavailability of free oxylipins were quantified using Reverse-Phase Ultra- Performance Liquid Chromatography coupled to negative Electrospray Ionization and Triple Quadrupole Tandem Mass Spectrometry (RP-UPLC-ESI(-)-QqQ-MS/MS) in Npc1 knock-in (NPC1ki) and Wildtype (WT) mice. Total cholesterol and fatty acids including polyunsaturated fatty acid (PUFA) precursors to oxylipins, were quantified using Gas Chromatography coupled to Flame Ionization Detection (GC-FID). Compared to WT mice, female NPC1ki mice, but not males, exhibited significantly elevated levels of free pro-resolving fatty acid epoxides (EpETrE and EpDPE) from the cytochrome P450 (CYP) pathway. Esterified mono- and dihydroxy lipid mediators derived from the lipoxygenase (LOX) and soluble epoxide hydrolase (sEH) pathways were mainly increased in NPC1ki females, suggesting enhanced sequestration of pro-inflammatory LOX and sEH metabolites. While PUFAs and cholesterol concentrations were not significantly different between groups, myristic (C14:0) and palmitoleic acid (C16:1n-7) were significantly elevated in female NPC1ki mice compared to WT controls. These findings suggest sex-specific adaptations in inflammation resolution pathways in NPC, with females exhibiting distinct inflammatory responses that may drive sex-related differences in disease pathogenesis. Our findings underscore the need for sex-specific therapeutic approaches to improve NPC treatment outcomes.

Animals

Characterization of the purified components of a new homologous series of alpha-mycolic acids from Mycobacterium tuberculosis H37Ra.

Homologous series of alpha-mycolic acids from Mycobacterium tuberculosis H37Ra were separated according to size as their p-bromophenacyl ester by bonded C18 reverse-phase, high performance liquid chromatography. Mass spectrometry of the prominent components separated by high performance liquid chromatography gave relatively simple spectra and showed a series of components differing by 28 atomic mass units. A total of different structures were identified. The structures were established as follow: (formula see text) where a = 17, 18, 19; b = 10; c = 15, 17, 19, 21; and d = 21, 23. The C76 and C78 acids contained some C24 alpha-branch acid (d - 21), whereas the C80 and C82 acids contained some a = 19 acids. Several new homologous series were revealed.

Chromatography, High Pressure Liquid

Acyclovir kinetics after intravenous infusion.

The disposition and safety of the antiviral drug acyclovir were studied in 14 subjects with advanced malignancies. Acyclovir was administered by a 1-hr intravenous infusion at doses of 0.5, 1.0, 2.5, and 5.0 mg/kg. At the end of infusion, mean peak plasma levels (+/- SEM), determined by radioimmunoassay, were 6.4 +/- 0.7, 12.1 +/- 2.3, 14.9 +/- 2.7, and 33.7 +/- 7.1 microM. The plasma concentration-time profiles could be described by a biexponential equation. The half-life of acyclovir in the slow disposition phase ranged from 2.2 to 5 hr and the drug was detected in the plasma for at least 18 hr after infusion. The total body clearance ranged from 117 to 396 ml/min/1.73 m2. A proportionality between area under the curve and dose suggests that acyclovir exhibits dose-independent kinetics in the dose range studied. There was wide variation in cumulative urinary excretion of unchanged drug, ranging from 30 to 69% of the dose. From renal clearances of acyclovir, which were higher than creatinine clearances, it appears that both glomerular filtration and tubular secretion contribute to its renal excretion. Analysis of the urine by reverse-phase high-performance liquid chromatography revealed the presence of the metabolite 9-carboxymethoxymethylguanine. There was no indication of toxicity either clinically or from laboratory findings in any of the study subjects. This study demonstrates that in addition to selectivity and low toxicity, the kinetic profile and metabolic disposition of acyclovir make it an attractive candidate for therapy in a variety of herpes infections.

Aged

Proteomic profiling of the aqueous extract from the antennal gland of the Pacific white shrimp, Litopenaeus vannamei.

The antennal gland (AnG) of decapod crustaceans has been proposed as a potential source of bioactive molecules involved in chemical communication; however, its protein composition remains largely unexplored. Here, we present the first reference proteomic map of the aqueous extract from the antennal gland of the Pacific white shrimp Litopenaeus vannamei. Protein extracts from immature and mature females were analyzed using an integrated workflow combining one-dimensional SDS-PAGE, reverse-phase high-performance liquid chromatography (RP-HPLC), and nanoLC-tandem mass spectrometry. Electrophoretic and chromatographic analyses revealed a high degree of qualitative similarity between reproductive stages. SDS-PAGE resolved six major protein bands (&#x223c;227, 166, 77, 42, 35, and 17&#xa0;kDa), most comprising multiple co-migrating proteins as revealed by LC-MS/MS. Hemocyanin was identified as the predominant protein and was detected across several electrophoretic bands. Additional proteins were associated with innate immunity, including &#x3b2;-1,3-glucan-binding protein and coagulable hemolymph protein; reproductive processes, including vitellogenin, spermatogonial stem-cell renewal factor, farnesoic acid O-methyltransferase, estrogen sulfotransferase, and prostaglandin reductase 1; as well as energy metabolism, protein homeostasis, cytoskeletal organization, and intracellular trafficking. Because several identified proteins are widely distributed or known hemolymph components, their detection cannot be assumed to reflect AnG-specific expression or function. Collectively, these findings establish a molecular reference for the L. vannamei AnG and reveal protein components associated with multiple physiological processes. This dataset provides a proteomic framework for future comparative and functional studies aimed at elucidating antennal gland physiology and experimentally evaluating the potential involvement of proteinaceous or peptide-based molecules in chemical communication in decapod crustaceans.

Animals

5',8-cyclo-dAdo and 8-oxo-dAdo DNA Lesions Are Both Substrates of Adenosine Deaminase: A Preliminary Study.

Genetic information, whether inside or outside the nucleus, is exposed to a variety of harmful physico-chemical factors. Although DNA damage repair systems have been extensively studied, little information about post-repair and non-genomic DNA damage metabolism is available in the literature. Adenosine deaminase (ADA) is an abundant enzyme found on both sides of the cell membrane that regulates the concentration of adenine derivatives. In this article, it has been shown that 7,8-dihydro-8-oxo-2'-deoxyadenosine (OXOdAdo) and (5'R/S) 5',8-cyclo-2'-deoxyadenosine ((5'R/S)cdAdo) are suitable substrates for ADA. For this purpose, theoretical Density Functional Tight Binding and RP-HPLC analyses were applied. The products of ADA activity, i.e., OXOdIno (7,8-dihydro-8-oxo-2'-deoxyinosine) and (5'R/S) cdIno ((5'R/S) 8-cyclo-2'-deoxyinosine), were identified and confirmed by high-resolution mass spectroscopy. Although the (5'R) and (5'S)cdAdo enzymatic deamination processes are much slower (34% and 32% after 168 h, respectively) than the process observed for dAdo, 5',8-cyclo-2'-deoxyinosine should be considered when monitoring cyclopurine levels in physiological fluids. The same should be considered in the case of OXOdAdo, which is completely converted to OXOdIno within one minute and may therefore be less visible than OXOdGuo during mass spectroscopy analysis. Both these observations are important, given the abundance of 2'-deoxyadenosine on both sides of the cell membrane and its potential conversion into OXOdAdo and (5'R/S)cdAdo. They may also explain why the observed level of OXOdAdo is much lower than that of OXOdGuo in cells and physiological fluids, even though their difference in ionisation potential is only 0.25 eV. Future studies are needed to further investigate the metabolism of DNA damage and to identify the enzymes involved in nucleic acid biochemistry.

Adenosine Deaminase

Relationship between changes in the translational apparatus and actinomycin production in Streptomyces antibioticus.

As previously reported (G. H. Jones, 1975), transfer ribonucleic acids (tRNA's) and ribosomes from actinomycin-producing cultures of Streptomyces antibioticus show a decreased ability to function in aminoacylation and translation as compared with the corresponding components from younger cells. Further, specific changes in the isoacceptor patterns are revealed when tRNA's from actinomycin-producing cells are compared with those of younger cells by reverse- phase column chromatography. A specific glycyl-tRNA species is eliminated from the reverse-phase profile of tRNA's from actinomycin-producing S. antibioticus cells as compared with younger cells. Changes in isoacceptor patterns were also observed for the amino acids methionine, valine, phenylalanine, and leucine. Actinomycin synthesis was inhibited by growing S. antibioticus cells in the presence of alpha-methyl-DL-tryptophan. Inhibition of actinomycin synthesis reversed the changes in tRNA observed in normally grown control cultures, although it had no demonstrable effect on the growth of the cells. Thus, tRNA from 48-h-old, alpha-methyl-tryptophan-grown cells had amino acid acceptor activity that was equal to or greater than that of tRNA from 12-h-old, normally grown cells. Similarly, the reverse-phase chromatographic pattern for glycyl-tRNA's from 48-h-old, alpha-methyl-tryptophan-grown cells was identical to that of the glycyl-tRNA's from 12-h-old, normally grown cells. In contrast, the ability of ribosomes from 48-h-old, alpha-methyl-tryptophan-grown cells to function in polypeptide synthesis in vitro was essentially identical to that of 48-h-old, normally grown cells. Ribosomes from 12-h-old, normally grown cells were severalfold more active in in vitro polypeptide synthesis.

Bacterial Proteins

Analysis for indole compounds in urine by high-performance liquid chromatography with fluorometric detection.

We describe a chromatographic system involving a high-performance chemically-bonded reverse-phase column and fluorescence detection for measurement of indoles in urine. We controlled retention and selectivity by optimizing the methanol content and pH of the mobile phase. Six reference indoles were separated in less than 20 min; three 5-hydroxyindoles were eluted in less than 7 min. About 5-15 ng of aqueous solutions of these compounds can be detected. The combination of selectivity (from use of the chromatographic column) and fluorescence detection permitted analysis for five of the six indoles after a single urine-deproteinization step.

5-Hydroxytryptophan

Purification and analysis of the purity of NADH.

We developed an analytical reverse-phase high-performance liquid chromatographic procedure for rapid assessment of the purity of NADH. The method completely separates adenosine monophosphate and adenosine diphosphoribose from NADH. By use of this analytical technique we found that preparative chromatography on DEAE-cellulose gives NADH that is free from adenine nucleotides as well as other impurities that commonly are present in NADH. The absorbance ratio at 260 and 340 nm of the purified NADH in 1.8 mmol/liter ammonium carbonate is 2.261 +/- 0.002 (+/- 1 SD).

Bicarbonates

Metabolism of octanoate and its effect on glucose and palmitate utilization by isolated fat cells.

Octanoate is avidly incorporated into triglycerides by isolated rat adipocytes in the presence of glucose via direct esterification without prior beta-oxidation to acetyl CoA. This was shown by separation of the products formed from (1-14C) octanoate into lipid classes using Florisil columns, and after alkaline hydrolysis of the triglyceride fraction, by cochromatogrpahy with authentic fatty acids on reverse-phase Celite columns. The relative contribution of (U-14C) glucose and (1-14C) octanoate to triglyceride synthesis and CO2 formation were studied under a variety of conditions. Concentrations of octanoate below 0.5 mM have a stimulatory effect on the conversion of (U-14C) glucose to CO2, triglycerides and esterified fatty acids. However, a marked depression of fatty acid synthesis from (U-14C) glucose was observed in the presence of millimolar concentrations of octanoate. Octanoate had no effect on the esterification of palmitate, but palmitate strongly depressed the ability of rat adipocytes to esterify octanoate.

Adipose Tissue