Article pubs.acs.org/JAFC
Accurate Determination of Reference Materials and Natural Isolates by Means of Quantitative 1H NMR Spectroscopy Oliver Frank,*,† Johanna Karoline Kreissl,‡ Andreas Daschner,† and Thomas Hofmann† †
Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner-Strasse 34, D-85354 Freising-Weihenstephan, Germany ‡ German Research Center for Food Chemistry, Lise-Meitner-Strasse 34, D-85354 Freising-Weihenstephan, Germany S Supporting Information *
ABSTRACT: A fast and precise proton nuclear magnetic resonance (qHNMR) method for the quantitative determination of low molecular weight target molecules in reference materials and natural isolates has been validated using ERETIC 2 (Electronic REference To access In vivo Concentrations) based on the PULCON (PULse length based CONcentration determination) methodology and compared to the gravimetric results. Using an Avance III NMR spectrometer (400 MHz) equipped with a broad band observe (BBO) probe, the qHNMR method was validated by determining its linearity, range, precision, and accuracy as well as robustness and limit of quantitation. The linearity of the method was assessed by measuring samples of L-tyrosine, caffeine, or benzoic acid in a concentration range between 0.3 and 16.5 mmol/L (r2 ≥ 0.99), whereas the interday and intraday precisions were found to be ≤2%. The recovery of a range of reference compounds was ≥98.5%, thus demonstrating the qHNMR method as a precise tool for the rapid quantitation (∼15 min) of food-related target compounds in reference materials and natural isolates such as nucleotides, polyphenols, or cyclic peptides. KEYWORDS: quantitative NMR, qHNMR, reference compounds, ERETIC 2, PULCON
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INTRODUCTION Reference compounds isolated from natural sources (foods, botanicals, biofluids) or prepared by organic synthesis are often contaminated with byproducts and/or contain substantial amounts of water and inorganic buffer salts, respectively. The use of such isolates as suitable reference standards for targeted quantitative analysis by means of high-performance liquid chromatography (HPLC), gas chromatography (GC), or capillary electophoresis (CE) hyphenated with evaporative light scattering detector (ELSD), ultraviolet−visible (UV−vis), infrared (IR), and mass spectrometry (MS) detection, respectively, requires their reliable purity assessment as well as the precise determination of their balance weight or concentration in solution. However, coelution with structurally related impurities, lacking chromophoric system for UV−vis detection, matrix effects, and/or low ionization potential during MS detection, and high volatility limiting the use of an ELSD often hamper a selective and absolute quantitation of target analytes identified.1−3 Moreover, the quantitative determination of unknown isomers or structural analogues of a given target compound by means of UV−vis and MS detection, respectively, is not possible in a reliable way as their extinction coefficient and/or ionization potential might differ substantially from the reference compound used for calibration.3,4 Although showing a purity of >95% on the basis of NMR data, isolated compounds might contain significant amounts of water and/or inorganic salts, which are not detectable in the NMR spectrum. Furthermore, low amounts of target compounds (
