The binding sites and consecutive binding constants of alkali metal ions, (M ϩ ϭ Na ϩ , K ϩ , Rb ϩ , and Cs ϩ ), to thrombin-binding aptamer (TBA) DNA were studied by Fouriertransform ion cyclotron resonance spectrometry. TBA-metal complexes were produced by electrospray ionization (ESI) and the ions of interest were mass-selected for further characterization. The structural motif of TBA in an ESI solution was checked by circular dichroism. The metal-binding constants and sites were determined by the titration method and infrared multiphoton dissociation (IRMPD), respectively. The binding constant of potassium is 5-8 times greater than those of other alkali metal ions, and the potassium binding site is different from other metal binding sites. In the 1:1 TBA-metal complex, potassium is coordinated between the bottom G-quartet and two adjacent TT loops of TBA. In the 1:2 TBA-metal complex, the second potassium ion binds at the TGT loop of TBA, which is in line with the antiparallel G-quadruplex structure of TBA. On the other hand, other alkali metal ions bind at the lateral TGT loop in both 1:1 and 1:2 complexes, presumably due to the formation of ion-pair adducts. IRMPD studies of the binding sites in combination with measurements of the consecutive binding constants help elucidate the binding modes of alkali metal ions on DNA aptamer at the molecular level. . The active form of TBA adopts a chair-type intramolecular G-quadruplex (G4), where the two G-tetrads are interconnected through the lateral TT and TGT loops in antiparallel conformation [2][3][4][5][6]. Some metal ions induce the structural transition to the G4 structure upon binding [7][8][9][10]. The binding modes of metal ions on the G4 structure are of fundamental interest in understanding their specific and/or nonspecific interactions. Various methods have been applied to the studies of binding modes and structures of TBA-metal complexes. NMR experiments have shown that potassium induces the chair-type G-quadruplex formation, but sodium does not [11]. Circular dichroism (CD) and UV absorption studies have shown that potassium, strontium, and barium stabilize the G4 structure in solution [12]. Recently, electrospray ionization mass spectrometry (ESI-MS) has confirmed the unimolecular G4 structure of TBA-metal (metal ϭ potassium, strontium and barium) complexes in the gas phase by H/D exchange [13]. Here, we studied the binding modes of alkali metal ions on TBA and determined the consecutive binding constants to unravel the specific and/or non-specific interactions between alkali metal ions and G4 TBA.Most of the structural studies carried out in solution deal with an ensemble of dynamic structures present under ambient conditions. To more firmly establish the binding mode, we need to employ a molecular probe that is selective toward the specific ions of interest. ESI-MS offers a mass-specific means to bring the complex ions present in solution into the gas-phase [14,15] for the studies of noncovalent ligand-nucleic acid interactions. Moreover, ESI-MS h...

A certified reference material (CRM), KRISS CRM 108-10-003, has been developed for analysis of acrylamide in potato chips, as a representative of carbohydrate-rich food cooked in high-temperature oil. The material was prepared by grinding commercially available potato chips to a paste which was then homogenized, bottled in 15-g units, and stored at -70 °C. Certification, homogeneity and stability testing, were carried out by liquid chromatography-isotope-dilution mass spectrometry (ID-LC-MS). A single ID-LC-MS measurement was performed for each of 10 selected units for certification and homogeneity assessment. The mean measurement result for the 10 bottles, 0.455 ± 0.012 mg kg(-1), was assigned as the certified value of the CRM. The between-bottle homogeneity was 0.8% of the certified value. The within-bottle homogeneity, tested by measuring three replicate sub-samples from each of three randomly selected bottles, was similar to the between-bottle homogeneity. The stability of the CRM under storage conditions (-70 °C) was tested for 21 months and no change in the acrylamide content was observed within the measurement uncertainty. Stability of the CRM at -20 °C (storage at user's site) and room temperature (for regular use and transportation) was also tested. Also presented is the newly designed procedure for evaluating the uncertainty of the certified value for the characterization scheme used in this study.

We propose isotope-dilution mass spectrometry as a candidate reference method for determination of serum cortisol. The method uses liquid chromatography-mass spectrometry (LC-MS), interfaced with electrospray ionization, and selective monitoring of the [M + H]+ ions of cortisol and isotopically labeled cortisol. The isotope-dilution-liquid chromatography-mass spectrometry (ID-LC-MS) method simplifies sample-preparation, because samples are processed by simple solvent extraction without further clean-up and derivatization. We studied the time required for complete equilibration of endogenous cortisol and labeled cortisol spiked into serum and found it to be less than 1 h. The repeatability and the reproducibility of the method were evaluated and found to be 0.55% of the measurement value. CRM 192 and 193 from the Bureau Communautaire de Reference were analyzed for verification of the method. The results obtained from the ID-LC-MS method agreed with the certified values. The relative uncertainty of measurement results for samples in the range of a few tens of micrograms per kilogram to several hundred micrograms per kilogram was evaluated and found to be 0.56%. Immunoassay carried out by three independent clinical laboratories produced results more than 15% higher than this ID-LC-MS method, suggesting the presence of bias in the immunoassay methods.

A model system has been established for the evaluation of the uncertainty of the value from measurements of multiple subsamples by isotope dilution mass spectrometry (IDMS). In this report, we apply this model system for the evaluation of measurement uncertainty in determination of folic acid in infant formula. Five subsamples were analyzed by IDMS. The mean of the measurement results of the five subsamples was assigned as the final measurement value. The standard deviation (s) of the results from five subsamples was attributable to repeatability of the measurement. The uncertainty components in the IDMS measurement methods were categorized into two groups. Group I includes uncertainty components which give common systematic effects to all subsamples and do not contribute to the variation among multiple measurements (repeatability). Group II includes uncertainty components that give random effects on the measurement results, and are related with the measurement repeatability. These random effects are attributed to s. Therefore, the uncertainty of the final value was calculated by combining the standard deviation of the mean of multiple measurements, s/√n (where n = 5), and the measurement uncertainty associated with the uncertainty components that give systematic effects.

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