ERETIC2 (Electronic Reference To access In vivo Concentrations 2) based on PULCON (Pulse Length–based Concentration determination) methodology is a quantitative NMR (qNMR) using an external standard. The performance of the PULCON method was assessed using maleic acid (MA). Quantification of the diarrhetic shellfish toxin and okadaic acid by PULCON was successfully consistent with that obtained by a conventional internal standard method, demonstrating that the PULCON method is useful for the quantification of invaluable marine toxins without any contaminations by an internal standard.
The evaluation of seven internal standard reference materials (ISRMs) to act as a ‘universal’ SI-traceable calibrator suite for organic compound purity determination by quantitative nuclear magnetic resonance (qNMR) spectroscopy is described. The set of compounds demonstrated to constitute such a suite are: potassium hydrogen phthalate (KHP), maleic acid (MA), 3,5-bis-trifluoromethyl benzoic acid (BTFMBA), dimethyl sulfone (DMSO2), dimethyl terephthalate (DMTP), 1,4-bis-trimethlsilyl benzene (BTMSB or BTMSB-d
4) and perdeuterated sodium 3-trimethylsilyl-1-propanesulfonate (DSS-d
6). The compounds were selected such that at least one ISRM should be suitable for use as the internal standard for the qNMR purity assignment of an organic compound soluble in a given deuterated solvent. They allow for the selection for use as the internal reference for quantitative integration from a set of simple, sharp NMR signals dispersed over the proton chemical shift range.
Optimized conditions for acquiring qNMR spectra were developed and are described, as well as the results of an extensive series of studies validating the use of the ISRM suite to assign mass fraction values in four representative solvents (D2O, DMSO-d
6, CD3OD and CDCl3). Proper use and application of these ISRMs result in standard uncertainties in the assigned values of the analyte of interest of the order of 1 mg g−1 in optimal cases. These materials are of particular interest for the mass fraction purity determinations by qNMR of organic compound reference materials required as analyte specific calibrators to underpin the SI-traceability of the results for routine laboratory analysis based on techniques such as gas and liquid chromatography.
We have developed a novel amino acid analysis method using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups). The amino, carboxyl, and phenolic hydroxyl groups of the amino acids were derivatized with 1-bromobutane so that the hydrophobicities and basicities of the amino acids were improved. The derivatized amino acids, including amino group-modified amino acids, could be detected with high sensitivity using liquid chromatography/tandem mass spectrometry (LC-MS/MS). In this study, 17 amino acids obtained by hydrolyzing proteins and 4 amino group-modified amino acids found in the human body (N,N-dimethylglycine, N-formyl-L-methionine, L-pyroglutamic acid, and sarcosine) were selected as target compounds. The 21 derivatized amino acids could be separated using an octadecyl-silylated silica column within 20 min and simultaneously detected. The detection limits for the 21 amino acids were 5.4-91 fmol, and the calibration curves were linear over the range of 10-100 nmol L(-1) (r(2) > 0.9984) with good repeatability. A confirmatory experiment showed that our proposed method could be applied to the determination of a protein certified reference material using the analysis of 12 amino acids combined with isotope dilution mass spectrometry. Furthermore, the proposed method was successfully applied to a stable isotope-coded derivatization method using 1-bromobutane and 1-bromobutane-4,4,4-d3 for comparative analysis of amino acids in human serum.
We have applied a combination of H quantitative NMR spectroscopy (H-qNMR) and chromatography (GC or LC) to establish reliable analytical methods (qNMR/GC and qNMR/LC) for organic compounds. In this method, a reference standard is used as an internal standard for both H-qNMR and chromatography to estimate relative molar sensitivity (RMS) for analytes. The RMS values are calculated from the molar ratios between analytes and the reference standard obtained byH-qNMR; and the response ratio between them obtained by chromatography. Concentrations of analytes in the organic solution can be simultaneously determined from the RMS and amount of the reference standard added in the sample solution. This analytical method is an innovative one because only one reference standard with International System of Units (SI)-traceable property value, purity, or concentration, is necessary to determine accurate concentrations of multiple organic components in organic solutions, without the respective certified reference standards for various analytes. To verify this method, a certified reference material, NIST SRM 1647f, was used. Among the 16 polycyclic aromatic hydrocarbons (PAHs) included in NIST SRM 1647f, naphthalene and benzo[a]pyrene were selected as analytes for this method, using 1,4-bis(trimethylsilyl)benzene-d as the reference standard. Each quantitative value obtained by qNMR/GC and qNMR/LC agreed with each certified value within its expanded uncertainty.
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