title: Evaluation of a liquid chromatography method for compound-specific δ13C analysis of plant carbohydrates in alkaline media.
abstract: BACKGROUND
  Isotope analysis of carbohydrates is important for improved understanding of plant carbon metabolism and plant physiological response to the environment. High-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) for direct compound-specific δ(13)C measurements of soluble carbohydrates has recently been developed, but the still challenging sample preparation and the fact that no single method is capable of separating all compounds of interest hinder its wide-spread application. Here we tested in detail a chromatography method in alkaline media.
  
  
  METHODS
  We examined the most suitable chromatographic conditions for HPLC/IRMS analysis of carbohydrates in aqueous conifer needle extracts using a CarboPac PA20 anion-exchange column with NaOH eluent, paying specific attention to compound yields, carbon isotope fractionation processes and the reproducibility of the method. Furthermore, we adapted and calibrated sample preparation methods for HPLC/IRMS analysis. OnGuard II cartridges were used for sample purification.
  
  
  RESULTS
  Good peak separation and highly linear and reproducible concentration and δ(13)C measurements were obtained. The alkaline eluent was observed to induce isomerization of hexoses, detected as reduced yields and (13)C fractionation of the affected compounds. A reproducible pre-purification method providing ~100% yield for the carbohydrate compounds of interest was calibrated.
  
  
  CONCLUSIONS
  The good level of peak separation obtained in this study is reflected in the good precision and linearity of concentration and δ(13)C results. The data provided crucial information on the behaviour of sugars in LC analysis with alkaline media. The observations highlight the importance for the application of compound-matched standard solution for the detection and correction of instrumental biases in concentration and δ(13)C analysis performed under identical chromatographic conditions. The calibrated pre-purification method is well suited for studies with complex matrices that disable the use of a spiked internal standard for the detection of procedural losses.
authors.affiliation: Laboratory of Atmospheric Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland. katja.rinne@psi.ch
authors.forename: Katja T
authors.initials: KT
authors.lastname: Rinne
authors.forename: Matthias
authors.initials: M
authors.lastname: Saurer
authors.forename: Kathrin
authors.initials: K
authors.lastname: Streit
authors.forename: Rolf T W
authors.initials: RT
authors.lastname: Siegwolf
mesh_terms: D002138:Calibration; D002241:Carbohydrates / Q000032:analysis*; D002247:Carbon Isotopes / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D016014:Linear Models; D028201:Pinaceae / Q000737:chemistry*; D015203:Reproducibility of Results
chemical_list: D002241:Carbohydrates; D002247:Carbon Isotopes