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PubMed2006

Identification and characterization of isomeric intermediates in a catalyst formation reaction by means of speciation analysis using HPLC-ICPMS and HPLC-ESI-MS.

Information on chemical speciation is much needed in mechanistic and kinetic studies on catalyst formation processes in pharmaceutical research. Speciation analysis was applied to the identification and quantification of various rhodium species involved in a ligand exchange process leading to formation of catalyst dirhodium(II) tetrakis[methyl 2-oxopyrrolidin-5(S)-carboxylate]. Inductively coupled plasma mass spectrometry (ICPMS) was used as an element-specific detector following species separation by reversed-phase high-performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI-MS) was used for species identification and confirmation. A novel interface between the HPLC and ICPMS, which consisted of an eluent splitter, a desolvation unit, and the ICPMS built-in peristaltic pump, enabled the use of RP-HPLC with gradient elution and up to 100% organic components in the LC eluent without organic loading in the plasma. A variety of reaction intermediates were identified and quantified along the pathway to formation of the desired product, including isomeric di-, tri-, and tetrasubstituted species previously believed to be absent. This has provided new insights into the mechanism and kinetics of the reaction. The combination of HPLC-ICPMS and HPLC-ESI-MS has proven to be a valuable tool for the investigation of species evolution in catalyst formation process.

Catalysis↗

PubMed1996

High-performance liquid chromatography-electrospray mass spectrometry of retinoids.

High-performance liquid chromatography (HPLC)-electrospray mass spectrometry (LC-MS) was used to analyze vitamin A-active retinoids including retinoic acid, retinol, retinal, and retinyl acetate. Unlike previous LC-MS methods such as negative ion electron capture chemical ionization, no derivatization of retinoic acid was required. HPLC separations were carried out on a C30 reversed phase column with gradient elution using mobile phases containing water, methanol, and methyl-tert-butyl ether. Ammonium acetate (5 mM) was added to the mobile phase to facilitate ion pair formation during reversed phase HPLC of retinoic acid, and acetic acid (0.5% v/v) was added to the mobile phase to enhance protonation during LC-MS analysis of nonacidic retinoids. During negative ion electrospray, retinoic acid formed abundant deprotonated molecules, [M-H]-, of m/z 299 without significant fragmentation. Although retinol, retinal, and retinyl acetate did not ionize during negative ion electrospray, the positive ion electrospray mass spectra of these retinoids showed an abundant protonated molecule of m/z 285 for retinal and base peaks of m/z 269 corresponding to elimination of water or acetic acid from the protonated molecules of retinol or retinyl acetate, respectively. No ions from retinoic acid were detected during positive ion electrospray. Limits of detection for retinoic acid, retinal, retinol, and retinyl acetate were 23 pg, 1.0 ng, 0.5 ng, and 10 ng, respectively.

Diterpenes↗

PubMed1993

Chromatographic analysis and pharmacokinetics of liposome-encapsulated doxorubicin in non-small-cell lung cancer patients.

A sensitive and specific quantitative assay for total doxorubicin concentrations in plasma containing liposome-encapsulated doxorubicin hydrochloride (TLC D-99) was developed, with solvent extraction and reversed-phase high-performance liquid chromatography (HPLC). Separation of doxorubicin from its metabolites was accomplished with a 15 cm x 3.9 mm i.d., microBondapak phenyl analytical HPLC column. Optimum chromatographic conditions, obtained with a mobile phase gradient from 85 to 50% (v/v) 16 mM ammonium formate buffer in tetrahydrofuran at a flow rate of 2 mL/min, gave a detection limit of 0.3 pmol/injection. Eleven-point standard curves with from 0.00595 to 29.8 microM TLC D-99 and 0.1 microM internal standard in plasma were analyzed on three separate occasions to formally validate this assay. An overall correlation coefficient of 0.9985 was found for the logarithmic transformed data. The pharmacokinetic characteristics of doxorubicin were investigated after administration of TLC D-99 to 12 non-small-cell lung cancer patients as an intravenous infusion at doses of 60 and 75 mg/m2. The data are best described by a three-compartment model with alpha, beta, and gamma elimination half-lives of 0.0721, 2.84, and 25.2 h for the 60-mg/m2 group and 0.103, 2.56, and 14.9 h for the 75-mg/m2 patients. A mean plasma clearance of 9.89 L/h (range: 1.95 to 23.4 L/h) was found for the 60-mg/m2 patients, with that from the 75-mg/m2 group being within these values. Mean area under the plasma concentration versus time curve estimates of 37.1 and 47.9 microM/h were observed for the patients receiving 60 and 75 mg/m2, respectively. The plasma concentration-time course for total doxorubicin following administration of TLC D-99 suggests that the disposition of the liposomal formulation is determined more by the pharmacokinetics of the liposome than the encapsulated drug.

Adult↗

PubMed1988

HPLC enrichment of hydrophobic DNA--carcinogen adducts for enhanced sensitivity of 32P-postlabeling analysis.

The sensitivity of 32P-postlabeling analysis for the quantitation of DNA-carcinogen adducts can be sharply increased by enriching adducted nucleotides relative to normal nucleotides in digested DNA samples prior to postlabeling. Normal and adducted nucleotides from benzo[a]pyrene-adducted DNA were injected onto a reverse phase HPLC column. Normal nucleotides were eluted with 5% methanol, 95% 1 M ammonium formate, pH 3.5. Hydrophobic adducted nucleotides were then recovered from the column using either a linear gradient of methanol (to fractionate adducts) or a step gradient of methanol to elute all hydrophobic adducts in one fraction. Recovered chromatographic fractions were dried, postlabeled with 32P, and aromatic DNA--carcinogen adducts were detected and quantitated using TLC on polyethyleneimine cellulose. Results from the HPLC adduct enrichment procedure were generally similar to those from an alternative enrichment scheme utilizing nuclease P1 to selectively dephosphorylate normal nucleotides. HPLC and nuclease P1 procedures are easily combined for the dual analysis of DNA samples from organisms exposed to environmental carcinogens.

Adenosine Triphosphate↗

PubMed2006

Reaction product analysis by high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance Application to the absolute configuration determination of naturally occurring polyyne alcohols.

The absolute configuration of secondary hydroxy functions of seven natural occurring polyyne derivatives has been elucidated by the application of Mosher method of diastereomeric methoxy-2-trifluoromethyl-phenylacetyl (MTPA) ester formation. High-performance liquid chromatography with diode array detection (HPLC-DAD) of the reaction mixture using a water/acetonitrile gradient allowed monitoring the reaction progress. Coupling of high-performance liquid chromatography to solid-phase extraction combined with nuclear magnetic resonance (HPLC-SPE-NMR) was utilized to generate highly reproducible (1)H and (19)F NMR data needed as input for the absolute configuration determination based on the analysis of relative shift differences. Chromatographic peaks of reaction substrates and reaction products bearing less 10mug analyte were trapped on SPE cartridges with the aid of water as makeup solvent. Deuterated chloroform was used to elute and transfer the peak content from the SPE to the 60mul flow cell of a 500MHz NMR spectrometer. For each analyte (1)H NMR spectra were obtained within 15min. Additionally (19)F NMR spectra were recorded for selected analytes in the same timeframe. Based on the obtained NMR data, the absolute configuration of all polyynes under investigation was successfully designated.

Alkaloids↗

PubMed1999

HPLC method for the analysis of acetonides of ecdysteroids providing structural information on different vicinal diols

The polyhydroxy structure of ecdysteroids provides a basis for the formation of various derivatives including the acetonides that have been frequently prepared mainly in preparative work. Acetonides can be formed between vicinal diols in cis position such as positions C-2/C-3 and C-20/C-22 in 20-hydroxyecdysone, for example. We have modified an established procedure for the preparation of acetonides, employing a higher concentration of phosphomolybdic acid as a catalyst, and developed a reverse-phase HPLC method to analyze the products on a micro scale. The derivatization method is fast, taking minutes or less, and does not produce artifacts. Thirteen structurally diverse ecdysteroids were studied in detail concerning speed of acetonide formation, and the number and type of intermediate and end products. Mass spectrometry coupled to HPLC was employed to confirm and/or identify ecdysteroids and their acetonide derivatives. Analysis of the chromatographic profiles revealed that detailed information on vicinal diols of ecdysteroids can be derived from the differences in retention times (Deltar.t.)1 of products relative to the parent steroids on reverse-phase HPLC using a linear water/methanol gradient for elution. The specific Deltar.t. values characterize a product as a monoacetonide, a diacetonide, or a triacetonide. Furthermore, this chromatographic shift upon acetonide formation is significantly different among the various monoacetonides and, thus, specific for the site on the ecdysteroid molecule. The same seems to hold for diacetonides. As variation of the Deltar.t. values for a specific type of acetonide was rather small among the tested ecdysteroids, the results should provide reliable structural information. Muristerone A did not completely fit this common pattern. However, this may specify its uncommon ecdysteroid structure. The method has recently been successfully employed to solve the structures of new ecdysteroid metabolites. Arch. Copyright 1999 Wiley-Liss, Inc.

Journal Article↗

PubMed2005

Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase.

Measurement of adenosine deaminase (ADA) activity using spectrophotometric method presents problem, regarding the quantitative estimation of the substrate degradation and product formation, due to the closely apposed lambda(max) of the substrates, product and the inhibitor. The feasibility of applying reverse-phase HPLC technique, for studying adenosine deaminase-catalyzed reaction product and inhibition study was examined. We have drawn a comparison between the HPLC-based method over the corresponding spectrophotometric method. A gradient elution pattern was used to separate substrate (adenosine and deoxyadenosine), product (inosine and deoxyinosine) and standard adenosine deaminase inhibitor (erythro-9-(3-nonyl-p-aminobenzyl)-adenine) in the HPLC method. The product formation was quantitated by monitoring the absorbance at 260 nm with the progress of time. The limit of detection as well as the limit of quantification of the respective enzymatic product were found to be in nano molar (nM) range in the HPLC method. This study was also extended to monitor adenosine deaminase activity in different cancer cells of hematological origin. The HPLC-based method is found to be suitable for the quantitative estimation of adenosine deaminase-catalyzed reaction product and for studying inhibition mechanism of different inhibitors. The HPLC-based method has specific advantages over the spectrophotometric method. Moreover, the concentration of different nucleotides in cell lysate and body fluid can be measured using this HPLC method.

Adenosine Deaminase↗

PubMed1999

Determination of a 'GW cocktail' of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry.

A mass spectrometry based method for the simultaneous determination of an in vivo Greenford-Ware or 'GW cocktail' of CYP450 probe substrates and their metabolites in both human plasma and urine is described. The probe substrates, caffeine, diclofenac, mephenytoin, debrisoquine, chlorzoxazone and midazolam, together with their respective metabolites and stable isotope labelled internal standards, are simultaneously extracted from the biological matrix using solid phase extraction in 96-well microtitre plate format, automated by means of a custom built Zymark robotic system. The extracts are analysed by fast gradient high performance liquid chromatography (HPLC) with detection by tandem mass spectrometry (MS/MS) using thermally and pneumatically assisted electrospray ionisation in both positive and negative ion modes and selected reaction monitoring. The methods are specific, accurate and precise with intra- and inter-assay precision (%CV) of less than 15% for all analytes.

Automation↗

PubMed2004

A proteome strategy for fractionating proteins and peptides using continuous free-flow electrophoresis coupled off-line to reversed-phase high-performance liquid chromatography.

Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from approximately 10(6) for cells to approximately 10(10) for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by approximately 0.02-0.10 pH unit depending on the gradient created, followed by rapid (approximately 6 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plots protein loci, pI in the first dimension and relative hydrophobicity (i.e., RP-HPLC retention time) in the second dimension. By coupling a diode-array detector in line with a multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by both UV absorbance and intrinsic fluorescence simultaneously from a single experiment. Triplicate analyses of standard proteins using a pH 3-10 gradient conducted over a 3-day period revealed a high system reproducibility with a SD of 0.57 (0.05 pH unit) within the FFE pools and 0.003 (0.18 s) for protein retention times in the second-dimension RP-HPLC step. In addition, we demonstrate that the FFE-IEF/RP-HPLC separation strategy can also be applied to complex mixtures of low molecular weight compounds such as peptides. With the facile ability to measure the pH of the isoelectric focused pools, peptide pI values can be estimated and used to qualify peptide identifications made using either MS/MS sequencing approaches or pI discriminated peptide mass fingerprinting. The calculated peak capacity of this 2D liquid-based FFE-IEF/RP-HPLC system is 6720.

Chromatography, High Pressure Liquid↗

PubMed1984

Evidence for intracellular formation of angiotensins: coexistence of renin and angiotensin-converting enzyme in Leydig cells of rat testis.

Leydig cells were purified from rat testes by discontinuous metrizamide density gradient and were shown to contain renin (EC 3.4.99.1), angiotensin-converting enzyme (dipeptidyl carboxypeptidase, (EC 3.4.15.1), and the peptide hormone angiotensins I, II and III as determined by the combined HPLC and radioimmunoassay. In germinal cells only angiotensin II (AII) was found at a significant level. These findings provide evidence for intracellular formation of AII in testicular cells and demonstrate that an intracellular renin-angiotensin system exists in normal non-transformed cells.

Angiotensins↗

PubMed1990

Determination of biopolymer (protein) molecular weights by gradient elution, reversed-phase high-performance liquid chromatography with low-angle laser light scattering detection.

The determination of molecular weights for certain proteins has been performed. This has involved the on-line coupling of gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A new 1.5-micron, non-porous, Monosphere RP-C8 column has been used in order to perform fast and conventional RP-HPLC gradients (5-45 min). Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for ribonuclease A, lysozyme, and bovine serum albumin. Standard mixtures of known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data have been obtained for all three proteins, but only under certain, conventional reversed-phase gradient elution conditions. Between 5-10 min of fast gradient elution, each protein appears to exhibit unusual Mw values, suggestive of aggregate formations. Methods have been developed to define the nature of such aggregates. The on-line coupling of modern RP-HPLC for biopolymers with LALLS represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.

Acetonitriles↗

PubMed2006

Simultaneous analysis of eight bioactive compounds in Danning tablet by HPLC-ESI-MS and HPLC-UV.

A high performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ESI-MS) and ultraviolet detector (UV) has been developed for the simultaneous analysis of eight bioactive compounds in Danning tablet (including hyperin, hesperidin, resveratrol, nobiletin, curcumine, emodin, chrysophanol, and physcion), a widely used prescription of traditional Chinese medicine (TCM). The chromatographic separation was performed on a ZORBAX Extend C(18) analytical column by gradient elution with acetonitrile and formate buffer (containing 0.05% formic acid, adjusted with triethylamine to pH 5.0) at a flow rate of 0.8 ml/min. The eight compounds in Danning tablet were identified and their MS(n) fractions were elucidated by using HPLC-ESI-MS, and the contents of these compounds were determined by using HPLC-UV method. The standard calibration curves were linear between 5.0 and 100 microg/ml for hyperin, 10-200 microg/ml for hesperidin, 1.0-150 microg/ml for resveratrol, 2.0-120 microg/ml for nobiletin, 2.0-225 microg/ml for curcumine, 20-300 microg/ml for emodin, 2.0-200 microg/ml for chrysophanol, and 20-250 microg/ml for physcion with regression coefficient r(2)>0.9995. The intra-day and inter-day precisions of this method were evaluated with the R.S.D. values less than 0.7% and 1.3%, respectively. The recoveries of the eight investigated compounds were ranged from 99.3% to 100.2% with R.S.D. values less than 1.5%. This method was successfully used to determine the 8 target compounds in 10 batches of Danning tablet.

Calibration↗

PubMed2006

On-line formation, separation, and estrogen receptor affinity screening of cytochrome P450-derived metabolites of selective estrogen receptor modulators.

We have developed a fully automated bioreactor coupled to an on-line receptor affinity detection system. This analytical system provides detailed information on pharmacologically active metabolites of selective estrogen receptor modulators (SERMs) generated by cytochromes P450 (P450s). We demonstrated this novel concept by investigating the metabolic activation of tamoxifen and raloxifene by P450-containing pig and rat liver microsomes. The high resolution screening (HRS) system is based on the coupling of a P450-bioreactor to an HPLC-based estrogen receptor alpha (ERalpha) affinity assay. P450-derived metabolites of the SERMs were generated in the bioreactor, subsequently trapped on-line with solid phase extraction, and finally separated with gradient HPLC. Upon elution, the metabolites were screened on affinity for ERalpha with an on-line HRS assay. With this HRS system, we were able to follow, in a time-dependent manner, the formation of ERalpha-binding metabolites of tamoxifen and raloxifene. By analyzing the bioaffinity chromatograms with liquid chromatography-tandem mass spectrometry, structural information of the pharmacologically active metabolites was obtained as well. For tamoxifen, 15 active and 6 nonactive metabolites were observed, of which 5 were of primary, 10 of secondary, and 6 of an as yet unknown order of metabolism. Raloxifene was biotransformed in three primary and three secondary metabolites. MS/MS analysis revealed that three of the observed active metabolites of raloxifene were not described before. The present automated on-line HRS system coupled to a P450-containing bioreactor and an ERalpha-affinity detector proved very efficient, sensitive, and selective in metabolic profiling of SERMs.

Animals↗

PubMed1989

Leukotriene formation by human polymorphonuclear leukocytes from endogenous arachidonate. Physiological triggers and modulation by prostanoids.

Human polymorphonuclear leukocytes (PMN) were isolated from freshly drawn venous blood by Dextran sedimentation and discontinuous Percoll gradient centrifugation. The effects of several putative triggers of the leukotriene formation such as C5a, PAF, FMLP, C3a, PMA, LTC4, LTD4, LTB4 or arachidonate were studied by RP-HPLC analysis. 280 nM C5a, 100 nM FMLP, 1 microM PAF or 20 microM arachidonate induced a marginal formation of 1.5-18 ng of LTB4 plus LTB4 metabolites/2 x 10(7) PMN. 560 nM C3a, 100 nM PMA, 1 microM LTC4, 1 microM LTD4 and 1 microM LTB4 each failed to induce any formation of 5-lipoxygenase products. Pretreatment of the cells with 40 microM ethylmercurithiosalicylate (merthiolate) enhanced the leukotriene formation by 100 nM FMLP about 40-fold, by 280 nM C3a about 120-fold and by 1 microM PAF about 14-fold. Merthiolate itself induced no leukotriene formation from human PMN and reduced the leukotriene formation by 20 microM arachidonate. The FMLP/merthiolate-induced activation of the PMN was concentration-dependent in respect to both FMLP and merthiolate. 1 microM LTC4, 1 microM LTD4 or 1 microM LTB4 also failed to trigger any LTB4 formation of merthiolate-treated PMN. 560 nM C3a or 100 nM PMA in combination with 40 microM merthiolate induced a slight formation of 28 ng and 10 ng of LTB4 plus LTB4 metabolites, respectively. The FMLP/merthiolate-induced leukotriene formation was modulated by prostanoids. PGE2, PGE1, PGD2 and 6-keto-PGE1 each evoked a concentration-dependent inhibition of the leukotriene formation with IC50 values of 0.07 microM, 0.18 microM, 0.27 microM and 6 microM respectively. In addition, significant inhibitory effects by PGI2, Iloprost (a carbacyclin analogue of prostacyclin), PGF2a or 6-keto-PGF1a were achieved; the corresponding IC50 values, however, amounted to 19-59 microM. Thus these compounds were about 500-fold less potent in comparison with PGE2 in inhibiting LTB4 formation by human PMN.

Arachidonic Acids↗

PubMed2005

Purification of sulforaphane from Brassica oleracea seed meal using low-pressure column chromatography.

Sulforaphane is an isothiocyanate that is present naturally in widely consumed Brassica oleracea vegetables and has been shown to block the formation of tumors. The contents of sulforaphane in five groups of B. oleracea seeds (broccoli, Brussels sprouts, cabbage, cauliflower and kale) were determined by RP-HPLC using linear gradient of acetonitrile in water. A new low-cost method to isolate and purify natural sulforaphane from B. oleracea seed meal was described in this work. Crude sulforaphane was first separated from B. oleracea seed meal by using immiscible solvent extraction with ethyl acetate, 10% ethanol and hexane, and the crude sulforaphane was used as raw materials to prepare high purity sulforaphane by low-pressure column chromatography of silica gel (200-300 mesh) with different eluents and elution modes. Compared with these different elution methods, the gradient elution was preferable to the isocratic elution for reducing the elution time and the eluent consumption and increasing the purity of sulforaphane product. The purity and recovery of sulforaphane were more than 90% in gradient elution.

Anticarcinogenic Agents↗

PubMed1990

[Effect of monoclonal antibody against methylcholanthrene-induced cytochrome P-450 forms on benzo(a)pyrene metabolism in hepatic microsomes of C57BL/10 mice].

In this study, hepatic microsomes from 5,6-benzoflavone induced C57BL/10 mice were used. To inhibit monooxygenase activities, the monoclonal antibody MAb 1-7-1 recognizing two isoenzymes of methylcholanthrene-induced cytochrome P-450 was applied. Microsomes were incubated with tritium labeled benzo(a)pyrene [G-3H]BP for 10 min at 37 degrees C. The incubation mixture contained: 50 mM potassium phosphate buffer, pH 7.25; 30 mM KCl; 3 mM MgCl2; 2 mM NADPH; 80 microM [G-3H]BP (specific activity 50 mCi/mmol); and monoclonal antibody MAb 1-7-1 or ascites fluid (NBS) containing nonspecific IgG as a control. The ratio of antibody protein/microsomal protein was 2:5. BP metabolites were extracted from incubation mixtures by ethyl acetate. The organic layer was dried over sodium sulfate, and evaporated under a stream of nitrogen. To separate BP metabolites HPLC technology was used. The column was eluted with methanol gradient (60-100%) for 45 minutes. The radio-activity of collected samples was determined using liquid scintillation counter. Differential inhibitory effects of MAb 1-7-1 on BP-metabolites formation were found, e.g. 7,8-diol was inhibited by 86.1% and quinones by 62.5%. The predominant metabolite, 3-OH-BP, was inhibited by 80.4%. Moreover, it was found that MAb 1-7-1 inhibition of benzo(a)pyrene hydroxylase activity (by 75.8%, as measured by fluorescent technique) was very similar to the inhibition of 3-OH-BP along with 9-OH-BP formation (as measured by HPLC).

Animals↗

PubMed2006

Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase.

Human estrogen sulfotransferase (SULT1E1) is involved in the regulation of 17beta-estradiol responsiveness and is believed to protect peripheral tissues from excessive estrogenic effects. Several assays already have been developed to investigate the inhibitory effect of endocrine-disrupting compounds (EDCs) on SULT1E1. However, most of these assays make use of the radiolabeled cofactor [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) or radiolabeled substrate [(3)H]estradiol. In this article, we describe the development and validation of an assay for the inhibition of human SULT1E1 that is rapid and simple and that uses the nonradioactive and noncarcinogenic 1-hydroxypyrene. A gradient HPLC separation of 15 min using a C18-RP column was developed to detect 1-hydroxypyrene and its metabolite pyrene 1-sulfate fluorescently. Time- and protein-dependent formation of pyrene 1-sulfate was investigated, and enzyme kinetics was determined (K(m)=6.4+/-0.8 nM and V(max)=158+/-19 pmol/min/microg SULT1E1). At higher 1-hydroxypyrene concentrations, the assay displayed non-Michaelis-Menten kinetics involving substrate inhibition. IC(50) values have been determined for eight known SULT1E1 inhibitors or competing substrates (17beta-estradiol, 17alpha-estradiol, genistein, 17alpha-ethynylestradiol, estrone, diethylstilbestrol, estriol, and hexestrol) and two previously unknown SULT1E1 inhibitors (zearalenone and dienestrol). The method was demonstrated to be easy, feasible, and highly reproducible for SULT1E1 screening assay inhibition studies.

Chromatography, High Pressure Liquid↗

PubMed2006

Analysis of phospholipid species in rat peritoneal surface layer by liquid chromatography/electrospray ionization ion-trap mass spectrometry.

The main phospholipids in rat peritoneal surface layer were analyzed by normal-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion-trap mass spectrometry (MS). By using a silica gel column and a gradient of hexane/isopropanol/water as mobile phase containing 5 mmol/L ammonium formate as modifiers, a baseline separation of glycerophosphoehtanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and lyso-phosphatidylcholine (LPC) was obtained and more than 90 phospholipid constituents in rat peritoneal surface were identified and determined by on-line ion-trap MS detection. The major ethanolamine glycerophospholipids in rat peritoneal surfaces were plasmalogens that were highly enriched in polyunsaturated fatty acids at the sn-2 position. In addition, the fragmentation patterns for each phospholipid class by the ion-trap MS were discussed.

Animals↗