Thermo Scientific Hypercarb Columns

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C LINICALApplication NotesFigure 1: LC-MS/MS traces showing chromatographic separation of(1) Hydroxy-proline; (2) Valine; (3) Leucine; (4) Allo-isoleucine; (5) Isoleucine.(A) Total ion chromatogram; (B) SRM chromatogram for isobaric compoundsof m/z 188; (C) SRM chromatogram for valine.Figure 2: Differenciation of isoleucine and allo-isoleucine from isobaric leucine:SRM chromatogram for transition 188.09 – 69.10 at 32 eV collision energy.No peak for leucine is present.ConclusionAn eight minute method is demonstrated for the analysis ofleucine and isomers. Chromatographic resolution of theseunderivatised amino acids, valine and hydroxyproline isachieved on a porous graphitic carbon column. The sensitivityof the method could be improved by reducing the columninternal diameter and therefore reduce both the split ratiointo the MS and sample dilution in the column. Futurework should also include the investigation of the effect ofchanging the electronic modifier (nonafluoropentanoic acid)and investigate its effect on chromatographic resolution andmethod sensitivity.References1. P. Chaimbault, K. Petritis, C. Elfakir, M. Dreux, J. Chromatogr. A, 2000,896, 253-26334
Determination of Occupational Exposure to Toluene,Xylene and Styrene Through Metabolite MonitoringUsing Isocratic HPLCLuisa Pereira, Brid Brosnan, Charlotte Blythe, Thermo Fisher Scientific, Runcorn, UKC LINICALIntroductionToluene, xylene and styrene are widely used as an industrialfeedstock and in the modern scientific workplace. Styrene isthe precursor compound to polystyrene. Toluene is commonlyused in the paint, plastics and printing industries. Xylene isconsumed in the leather, printing and rubber industry.Exposure to styrene, toluene or xylene has a detrimentaleffect upon the central nervous system. At low concentrations,symptoms of dizziness, headache, nausea and vomiting areexperienced together with respiratory tract irritation.Exposure can cause in-coordination and mental confusionand, as the central nervous system becomes further depressed,can result in unconsciousness and death. The monitoring andevaluation of the biological effects upon health of workersexposed to such chemicals is, therefore, vitally important.Traditionally, exposure to these chemicals has beenevaluated though measurement of their concentration inambient air where workers are more likely to be exposed to,however, it has been found in recent years that biologicalmonitoring (in blood or urine) affords a far more accurateestimate of exposure. For example, in the presence ofethanol, the adsorption of toluene into the blood is doublethat which occurs with exposure to toluene alone, thusillustrating that measurement of air concentration canresult in misleading exposure estimates. 1Upon inhalation/absorption into the body, all threechemicals undergo biological transformation. The metabolicreaction rates are fast, consequently, the products are rapidlyexcreted from the body. As such, levels of compounds inblood are low, thus the concentration of metabolites inurine is measured. The main metabolites are hippuric acid,o-, m-, p-methylhippuric acid, mandelic acid and phenylglyoxylicacid. These metabolites are polar, and isomeric innature. Consequently, Thermo Scientific Hypercarb (porousgraphitic carbon, PGC) presents itself as the ideal phasefor the separation of such analytes. Indeed Schlatter et alhighlighted the advantageous positional isomer separationperformance of Hypercarb with respect to the analysis ofcresol isomers in the urine of workers exposed to toluene. 2As hippuric acid is present in the urine of individuals whohave not been subject to toluene exposure, some authorsbelieve that measurement of cresols provides a validalternative to the monitoring of hippuric acid.This application note highlights the use of Hypercarbfor the effective separation of the associated compounds ofstyrene, toluene and xylene metabolism. Using a simple twocomponent isocratic mobile phase all major metabolites(and an internal standard) are separated in under 11 minutes.GoalTo detail the effective monitoring of excreted toluene,xylene and styrene metabolites using a PGC stationaryphase in isocratic mode.ExperimentalLC MethodColumn: Hypercarb 3 µm, 100 x 4.6 mm(part number 35003-104630)Mobile Phase: H 2 O / MeOH (50:50) +0.1% TFAFlow Rate: 1 mL/minTemperature: 25 °CDetection: UV at 240 nmInjection Volume: 10 µLResults and DiscussionThe separation achieved for the biological metabolites usingthe conditions described above is illustrated in Figure 1.The analysis is achieved in less than eleven minutes withall analytes possessing good peak shape (greatest asymmetryat 10% value measured for mandelic acid at 1.18).Figure 1: Metabolite separation: 1. Mandelic acid; 2. o-Methylhippuric acid;3. Hippuric acid; 4. Phenyl-gyloxylic acid; 5. p-Hydroxybenzoic acid (internalstandard); 6. m-Methylhippuric acid; 7. p-Methylhippuric acid.Application Notes35
Page 1 and 2: Applications NotebookIssue 1, June
Page 3 and 4: Applications ReviewApplication Chro
Page 5 and 6: Retention Mechanisms on PGCOn a mol
Page 7 and 8: Porous Graphitic Carbon for the LC/
Page 9 and 10: the retention mechanism is a combin
Page 11 and 12: Data ProcessingThermo Scientific Xt
Page 13 and 14: Type of LC ColumnPeptides Graphite
Page 15 and 16: Higher-charged 4+ metal-adducted pr
Page 17 and 18: The Use of Porous Graphitic Carbon
Page 19 and 20: The lowest concentration from the d
Page 21 and 22: The experimental conditions for the
Page 23 and 24: Fast LC Separation of Triazine Herb
Page 25 and 26: SamplesSamples of surface water (Sa
Page 27 and 28: Analysis of Polar Metabolites of At
Page 29 and 30: Compounds Log Kow Recovery (%) S.DA
Page 31 and 32: ExperimentalSample PreparationFor t
Page 33: Determination of Leucine and its Is
Page 37 and 38: Porous Graphitic Carbon for Inorgan
Page 39 and 40: F OOD AND B EVERAGEFigure 4: Column
Page 41 and 42: Application Area Analyte Group Refe
Page 43 and 44: 69. J. Bessarda, P. Saviuc, Y. Chan
Page 45 and 46: Aflatoxinsafla1Nonylphenol Isomers1
Page 47 and 48: Ordering InformationHypercarb Colum

Thermo Scientific Hypercarb Columns

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