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Chromatographic Conditions (chromatographic + condition)
Selected AbstractsA new metabolite of nodakenetin by rat liver microsomes and its quantification by RP-HPLC methodBIOMEDICAL CHROMATOGRAPHY, Issue 2 2010Peng Zhang Abstract The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital-pretreated rats and detected by reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3,(R)-hydroxy-nodakenetin-3,-ol and 3,(S)-hydroxy-nodakenetin-3,-ol, respectively, on the basis of their 1H-NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP-HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C18 column, a mobile phase with MeOH-H2O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat-inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50,400, 20,120 and 150,24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI. Copyright © 2009 John Wiley & Sons, Ltd. [source] Modeling of protein breakthrough performance in cryogel columns by taking into account the overall axial dispersionJOURNAL OF SEPARATION SCIENCE, JSS, Issue 15-16 2009Junxian Yun Abstract A model considering the overall axial dispersion for describing protein adsorption and breakthrough in monolithic cryogel beds has been developed. The microstructure of cryogels was characterized by tortuous capillaries with a normal diameter distribution but a constant pore wall thickness. The axial dispersion within cryogel columns was described by using the overall axial dispersion coefficient, which can be easily obtained by matching the experimental breakthrough curves without adsorption or measuring residence time distributions (RTDs). Experimental breakthrough curves of lysozyme within a metal-chelated affinity cryogel by Persson et al. (Biotechnol. Bioeng. 2004, 88, 224,236) and a cation-exchange cryogel by Yao et al. (J. Chromatogr. A 2007, 1157, 246,251) were employed as examples to test the model. The results showed that by using the axial dispersion coefficient and assuming uniform radial concentration profile at a given cross-section of the cryogel along the bed height, the model can describe the detailed behaviors of the in-bed overall axial dispersion, the in-pore mass transfer, as well as the protein adsorption and breakthrough. For a known overall axial dispersion coefficient, the lumped parameter of the mass transfer coefficient between the bulk liquid and the capillary wall can be determined by fitting the protein breakthrough curve at a known chromatographic condition. Once this parameter is determined, the model can be used to predict the protein breakthrough profiles under different conditions based on the basic physical parameters of the cryogel bed and the properties of the fluid and protein. The effective capillary diameters employed in the model are close to the actual pore sizes observed from the images by SEM. The model predictions of lysozyme breakthrough profiles at various flow rates are also in good agreement with the experimental data in both the metal-chelated affinity and cation-exchange cryogel columns. [source] Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes,JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2006Kyoungju Choi Abstract The metabolism of chlorpyrifos (CPS) and chlorpyrifos oxon (CPO) by human hepatocytes and human liver S9 fractions was investigated using LC-MS/MS. Cytochrome P450 (CYP)-dependent and phase II-related products were determined following incubation with CPS and CPO. CYP-related products, 3,5,6-trichloro-2-pyridinol (TCP), diethyl thiophosphate, and dealkylated CPS, were found following CPS treatment and dealkylated CPO following CPO treatment. Diethyl phosphate was not identified because of its high polarity and lack of retention with the chromatographic conditions employed. Phase II-related conjugates, including O- and S-glucuronides as well as 11 GSH-derived metabolites, were identified in CPS-treated human hepatocytes, although the O -sulfate of TCP conjugate was found only when human liver S9 fractions were used as the enzyme source. O -Glucuronide of TCP was also identified in CPO-treated hepatocytes. CPS and CPO were identified using HPLC,UV after CPS metabolism by the human liver S9 fraction. However, CPO was not found following treatment of human hepatocytes with either CPS or CPO. These results suggest that human liver plays an important role in detoxification, rather than activation, of CPS. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:279,291, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20145 [source] Pharmaceutical impurity identification: A case study using a multidisciplinary approachJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2004Karen M. Alsante Abstract A multidisciplinary team approach to identify pharmaceutical impurities is presented in this article. It includes a representative example of the methodology. The first step is to analyze the sample by LC-MS. If the structure of the unknown impurity cannot be conclusively determined by LC-MS, LC-NMR is employed. If the sample is unsuitable for LC-NMR, the impurity needs to be isolated for conventional NMR characterization. Although the technique of choice for isolation is preparative HPLC, enrichment is often necessary to improve preparative efficiency. One such technique is solid-phase extraction. For complete verification, synthesis may be necessary to compare spectroscopic characteristics to those observed in the original sample. Although not widely practiced, an effective means of getting valuable structural information is to conduct a degradation study on the purified impurity itself. This systematic strategy was successfully applied to the identification of an impurity in the active pharmaceutical ingredient 1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(1-hydroxy-1-methyl-ethyl)-furan-2-sulphonylurea. Identification required the use of all of the previously mentioned techniques. The instability of the impurity under acidic chromatographic conditions presented an additional challenge to purification and identification. However, we turned this acidic instability to an advantage, conducting a degradation study of the impurity, which provided extensive and useful information about its structure. The following discussion describes how the information gained from each analytical technique was brought together in a complementary fashion to elucidate a final structure. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:2296,2309, 2004 [source] Chiral stationary phases for separation of intermedine and lycopsamine enantiomers from Symphytum uplandicumJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2010Rahul S. Pawar Abstract Enantioseparation of the pyrrolizidine alkaloid isomers intermedine and lycopsamine, isolated from Symphytum uplandicum, is discussed. The separatory power of two immobilized carbohydrate-based chiral HPLC columns, Chiralpak IA and IC, in different chromatographic conditions is compared. The study demonstrated the importance of solvent and column selection while developing such chiral HPLC separation methods. The baseline HPLC separation of the two alkaloid isomers in preparatory scale is reported for the first time. The optimized separations were achieved on a Chiralpak IA column with mobile phases of ACN/methanol (80:20) and methanol/methyl- t -butyl ether (90:10), both containing 0.1% diethylamine. [source] Differentiation and dating of red ink entries of seals on documents by HPLC and GC/MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17 2009Ya-Tong Yao Abstract A novel approach for differentiation and dating of red ink entries of seals on documents was developed based on ion-pairing HPLC (IP-HPLC) and GC/MS. Sixty-nine red ink pastes of seals were collected and the chromatographic conditions for separation of the dye components by IP-HPLC and the volatile additives by GC/MS in the ink entries were optimized. According to the dye components and additives, the ink entries were classified by HPLC with a multi-wavelength UV detector. The volatile components of the inks were identified by GC/MS and the classification of the ink entries was also investigated based on these volatile additives. The results showed that most of the ink entries of the seals can be differentiated by combining HPLC with a multi-wavelength detector and GC/MS methods. The degradation of the standard dye mixtures and the compositional changes of the ink entries of seals were investigated in light or natural aging conditions. The results indicated that the dye components decomposed in light or natural storage conditions, while the rates of the degradation depended on the structures of the dye components, the aging conditions, even the additives of the ink pastes. The results also showed that there existed good relationships between the compositional changes of the ink entries and the aging time, which can provide scientific evidences and valuable clues for dating of the ink entries. [source] Assessing a novel microfluidic interface for shotgun proteome analysesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2007An Staes Abstract Microfluidic interfaces coupled to ESI mass spectrometers hold great potential for proteomics as they have been shown to augment the overall sensitivity of measurements and require only a minimum of operator manipulations as compared to conventional nano-LC interfaces. Here, we evaluated a new type of HPLC-Chips holding larger enrichment columns (thus an increased sample loading capacity) for gel-free proteome studies. A tryptic digest of a human T-cell proteome was fractionated by strong cation exchange chromatography and selected fractions were analyzed by MS/MS on an IT mass spectrometer using both the new HPLC-Chip as well as a conventional nano-LC-MS/MS interface. Our results indicate that the HPLC-Chip is capable of handling very complex peptide mixtures and, in fact, leads to the identification of more peptides and proteins as compared to when a conventional interface was used. The HPLC-Chip preferentially produced doubly charged tryptic peptides. We further show that MS/MS spectra of doubly charged tryptic peptide ions are more readily identified by MASCOT as compared to those from triply charged precursors and thus argue that besides the improved chromatographic conditions provided by the HPLC-Chip, its peptide charging profile might be a secondary factor leading to an increased proteome coverage. [source] Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea groupJOURNAL OF SEPARATION SCIENCE, JSS, Issue 7 2006Leila Kallel Abstract The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84 : 16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75 : 25 v/v) at a temperature not exceeding 20°C. This study reveals that the retention mechanism is ion-pairing. [source] High-performance liquid chromatographic resolution of 1-(1,4-benzodioxane-2-formyl)- piperazine enantiomers after chiral derivatizationJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2005Zhiqiong Chen Abstract Chiral separation of racemic mixtures is of the greatest importance to the pharmaceutical industry, as the isomers of a given racemate may exhibit substantially different pharmacological effects, not to mention possibly differing toxicity behaviour. A novel chiral separation method is developed for the determination of 1-(1,4-benzodioxane-2-formyl)piperazine (BFP) enantiomers. The indirect resolution is performed by applying precolumn derivatization with the chiral reagent 2,3,4,6-tetra- O -acetyl-,-D-glucopyranosyl isothiocyanate (GITC). The resulting diastereoisomers are separated on a reversed-phase ODS column with methanol-potassium dihydrogen phosphate (0.02mol/L, 50:50) as mobile phase. UV detection is at 250 nm. The effect of mobile phase composition upon resolution and analysis time is investigated. Two diastereoisomers show nearly base-line separation under optimal chromatographic conditions. The presented study provides a simple and accurate method for the enantiomeric quality control and the optical purity assay of BFP. [source] Application of hyphenated mass spectrometry techniques for the analysis of urinary free glucocorticoidsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2009Angela Cuzzola Alteration of levels of glucocorticoids in plasma and urine can be related to several diseases. In particular, the determination of endogenous glucocorticoids in urine has been reported to provide information on cortisol and cortisone status, on the activities of steroid hormone enzymes and on glucocorticoid metabolism. In this study, the application of hyphenated mass spectrometry techniques (GC/MS without derivatization and LC/MS) for the simultaneous analysis of free urinary cortisol (F), cortisone (E), tetrahydrocortisol (THF), allo-tetrahydrocortisol (A-THF) and tetrahydrocortisone (THE) was evaluated. A sample preparation protocol by solid-phase extraction, mass spectrometry parameters and chromatographic conditions for both techniques were carefully optimized in terms of extracting phase and solvents, matrix effects, recovery, sensitivity and compound resolution. Baseline separation was achieved for the five underivatized analytes both in GC and LC. The LC/MS/MS technique was more suitable for the analysis of urine samples, being less influenced by matrix effects and showing excellent sensitivity and selectivity. A preliminary application of the reported method for the diagnosis of metabolic diseases was also described. The determination of each analyte in its free form, described for the first time in the paper, offers new perspectives in the application of glucocorticoid analysis for diagnostic purposes. Copyright © 2009 John Wiley & Sons, Ltd. [source] Reduction of in-source collision-induced dissociation and thermolysis of sulopenem prodrugs for quantitative liquid chromatography/electrospray ionization mass spectrometric analysis by promoting sodium adduct formationRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2008Chad E. Wujcik Six chromatographically resolved sulopenem prodrugs were monitored for their potential to undergo both in-source collision-induced dissociation (CID) and thermolysis. Initial Q1 scans for each prodrug revealed the formation of intense [Prodrug2,+,H]+, [Prodrug2,+,Na]+, [Prodrug,+,Na]+, and [Sulopenem,+,Na]+ ions. Non-adduct-associated sulopenem ([Sulopenem,+,H]+) along with several additional lower mass ions were also observed. Product ion scans of [Prodrug3,+,Na]+ showed the retention of the sodium adduct in the collision cell continuing down to opening of the , -lactam ring. In-source CID and temperature experiments were conducted under chromatographic conditions while monitoring several of the latter ion transitions (i.e., adducts, dimers and degradants/fragments) for a given prodrug. The resulting ion profiles indicated the regions of greatest stability for temperature and declustering potential (DP) that provided the highest signal intensity for each prodrug and minimized in-source degradation. The heightened stability of adduct ions, relative to their appropriate counterpart (i.e., dimer to dimer adduct and prodrug to prodrug adduct ions), was observed under elevated temperature and DP conditions. The addition of 100,µM sodium to the mobile phase further enhanced the formation of these more stable adduct ions, yielding an optimal [Prodrug,+,Na]+ ion signal at temperatures from 400 to 600°C. A clinical liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for sulopenem prodrug PF-04064900 in buffered whole blood was successfully validated using sodium-fortified mobile phase and the [PF-04064900,+,Na]+ ion for quantitation. A conservative five-fold increase in sensitivity from previously validated preclinical assays using the [PF-04064900,+,H]+ precursor ion was achieved. Copyright © 2008 John Wiley & Sons, Ltd. [source] Separation of a BMS drug candidate and acyl glucuronide from seven glucuronide positional isomers in rat plasma via high-performance liquid chromatography with tandem mass spectrometric detectionRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2006Y.-J. Xue A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1- O - , glucuronide) in rat plasma. A 50-µL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C18, 3,mm,×,150,mm, 3,µm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10,min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000,ng/mL, was fitted to a 1/x2 weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide. Copyright © 2006 John Wiley & Sons, Ltd. [source] The factors that influence the elution order for the resolution of amino acids on vancomycin phase using the polar-organic mobile phases after their pre-column derivatization with electrophilic reagentsBIOMEDICAL CHROMATOGRAPHY, Issue 6 2005S. Chen Abstract A variety of amino acids were enantioresolved on a vancomycin bonded chiral phase using the polar-organic mobile phases after their pre-column derivatization with electrophilic reagents in alkaline medium. The resolution was highly dependent on the analyte's structure and was enhanced as the aromatic side-chain group on the skeleton of analyte for ,,, interaction with the chiral selector became available. The steric hindrance resulting from the bulky side-chain group on the analyte also affected the resolution. Elution reversal, not found on the teicoplanin phase under the same chromatographic conditions, was possible through altering the type of reagent used in the derivatizing reaction (e.g. 2,4-difluorophenylisothiocyanate to 2,4-difluorophenylisocyanate). It is believed that the steric hindrance, as a result of the bulky sulfur atom in reagents such as methylisothiocyanate and others examined in this study, was responsible for the reversed elution order. The bulkiness of the substituent on the aromatic ring of derivatizing reagents (i.e. 2,3- and 3,5-dichlorophenylisothiocyanate) was observed to affect the resolution and alter the elution order as well. Copyright © 2005 John Wiley & Sons, Ltd. [source] Determination of terbinafine hydrochloride in cat hair by two chromatographic methodsBIOMEDICAL CHROMATOGRAPHY, Issue 8 2001Jernej Kuz Terbinafine hydrochloride (terbHCl) concentration on the site of infection with Microsporum canis is a very important indicator of drug effectiveness. Several chromatographic methods exist that can be used for the determination of terbHCl concentration in biological samples. A high performance liquid chromatographic (HPLC) method and a gas chromatographic (GC) method have been compared and critically evaluated for the determination of a terbHCl levels in cat hair. The sensitivity and the linearity of the previously developed HPLC method were 0.25,ng/mL and 0.25,3000,ng/mL, respectively. The limit of quantification (LOQ) was 0.01,µg/g of terbHCl in cat hair, and reproducibility of 96.6% and recovery of 93.8% were achieved using appropriate sample pre-treatment and optimal chromatographic conditions. The sensitivity of the GC method, 25,ng/mL (LOQ 625 ppb), was much lower than that of the HPLC method. The GC method still enables determination of terbHCl in a range of concentrations in cat hair. The reproducibility of terbHCl for the cat hair samples was 95.3% and the recovery was only 70.0%. Both methods can be used for the evaluation of drug effectiveness in cats and both of them require only basic chromatographic equipment that can be found in most analytical laboratories. Copyright © 2001 John Wiley & Sons, Ltd. [source] Determination of catechin diastereomers from the leaves of Byrsonima species using chiral HPLC-PAD-CDCHIRALITY, Issue 8 2010Daniel Rinaldo Abstract When catechins are found in plant extracts, they are almost always identified as catechin and/or epicatechin probably due to stereoselectivity of the enzymes involved in the biosynthesis of these substances. However, the lack of reports regarding to ent -catechin as well as ent -epicatechin does not necessarily mean that these compounds have not been produced. In fact, most of the previous reports used chromatographic conditions not suitable for such separation. This article describes a simple and reliable analytical HPLC-PAD-CD method for simultaneous determination of catechin diastereomers both in infusions and extracts from the leaves of Byrsonima species. The direct separation of catechin, ent -catechin, epicatechin, and ent -epicatechin was obtained in normal phase by HPLC-PAD-CD using Chiralcel OD-H as chiral stationary phase and n -hexane/ethanol with 0.1% of TFA as mobile phase. Chirality, 2010. © 2010 Wiley-Liss, Inc. [source] Enantiomeric separation of imidazolinone herbicides using chiral high-performance liquid chromatographyCHIRALITY, Issue 3 2007Kunde Lin Abstract Chiral high-performance liquid chromatography (HPLC) is one of the most powerful tools to prepare enantiopure standards of chiral compounds. In this study, the enantiomeric separation of imidazolinone herbicides, i.e., imazethapyr, imazapyr, and imazaquin, was investigated using chiral HPLC. The enantioselectivity of Chiralpak AS, Chiralpak AD, Chiralcel OD, and Chiralcel OJ columns for the three analytes was compared under similar chromatographic conditions. Chiralcel OJ column showed the best chiral resolving capacity among the test columns. The resolved enantiomers were distinguished by their signs of circular dichroism detected at 275 nm and their structures confirmed with LC-mass spectrometric analysis. Factors affecting the chiral separation of imidazolinones on Chiralcel OJ column were characterized. Ethanol acted as a better polar modifier than the other alcohols including 2-propanol, 1-butanol, and 1-pentanol. Although the acidic modifier in the mobile phase did not influence chiral recognition, it was necessary for reducing the retention time of enantiomers and suppressing their peak tailing. Thermodynamic evaluation suggests that enantiomeric separation of imidazolinones on Chiralcel OJ column is an enthalpy-driven process from 10 to 40°C. This study also shows that small amounts of pure enantiomers of imidazolinones may be obtained by using the analytical chiral HPLC approach. Chirality 19, 2007. © 2006 Wiley-Liss, Inc. [source] | ||||||||||