A precise and rapid isotopomic analysis of small quantities of cholesterol at natural abundance by optimized 1H-13C 2D NMR

Abstract

Cholesterol, the principal zoosterol, is a key metabolite linked to several health complications. Studies have shown its potential as a metabolic biomarker for predicting various diseases and determining food origin. However, the existing INEPT (insensitive nuclei enhanced by polarization transfer) 13 C position-specific isotope analysis method of cholesterol by NMR was not suitable for very precise analysis of small quantities due to its long acquisition time and therefore is restricted to products rich in cholesterol. In this work, a symmetric and adiabatic heteronuclear single quantum coherence (HSQC) 2D NMR sequence was developed for the high-precision (few permil) analysis of small quantities of cholesterol. Adiabatic pulses were incremented for improving precision and sensitivity. Moreover, several strategies such as the use of non-uniform sampling, linear prediction, and variable recycling time were optimized to reduce the acquisition time. The number of increments and spectral range were also adjusted. The method was developed on a system with a cryogenically cooled probe and was not tested on a room-temperature system. Our new approach allowed analyzing as low as 5 mg of cholesterol in 31 min with a long-term repeatability lower than 2‰ on the 24 non-quaternary carbon atoms of the molecule comparing to 16.2 h for the same quantity using the existing INEPT method. This result makes conceivable the isotope analysis of matrices low in cholesterol.

Authentication of Agave Products through Isotopic Intramolecular 13C Content of Ethanol: Optimization and Validation of 13C Quantitative NMR Methodology

“…Especially, this approach could replace partial and destructive methods involving chemical cuts inside molecules followed by IRMS measurement of the resulting moieties for ethanol, 36 L-malic acid, 37 or ascorbic acid. 38 The use of 2D NMR pulse sequence 39 could also be a promising lead for additional sensitivity enhancement.…”

Quantitative NMR spectroscopy of complex mixtures

“…More recently, the sensitivity per unit time of this approach was further improved by the incorporation of adiabatic pulses and by the incorporation of a variable recycling time (VRT), opening the way to new applications on smaller sample amounts. 123 Although the analytical requirements and associated scientific questions are different, these developments are in synergy with those achieved in the field of metabolomics.…”

NMR-Based Method for Intramolecular 13C Distribution at Natural Abundance Adapted to Small Amounts of Glucose

“…3 To date, one of the major problems of PSIA is the amount of material required to carry out analyses. Nonpositional (also referred to as "global" hereafter) isotope analyses use isotope ratio monitoring mass spectrometry (irm-MS), with a typical sample size of a few milligrams for 13 C, 18 O, or 2 H analyses. 4 Even though valuable information can be retrieved from global analyses, PSIA is hardly accessible with irm-MS.…”

“…The usefulness of asHSQC for irm-NMR analysis has been demonstrated with the 13 C profile obtained on a few mg of cholesterol. 18 Such a performance was achieved due to (i) a high signal-to-noise ratio (SNR); (ii) incorporation of adiabatic and composite adiabatic 180°pulses for inversion and refocusing, respectively, improving reproducibility; 19 (iii) reduction of acquisition time by optimization of 2D-NMR parameters, via non-uniform sampling (NUS) 20 and variable recycling time (VRT); 21 and (iv) use of Shigemi tubes. As a result, a precision with a standard deviation lower than 3‰ was obtained in 31 min, using 5 mg of cholesterol.…”

“…The 1 H nucleus is observed, and since it has a much larger gyromagnetic ratio than 13 C, it facilitates quantitation of C-atoms to which a 1 H is attached. The usefulness of asHSQC for irm-NMR analysis has been demonstrated with the 13 C profile obtained on a few mg of cholesterol . Such a performance was achieved due to (i) a high signal-to-noise ratio (SNR); (ii) incorporation of adiabatic and composite adiabatic 180° pulses for inversion and refocusing, respectively, improving reproducibility; (iii) reduction of acquisition time by optimization of 2D-NMR parameters, via non-uniform sampling (NUS) and variable recycling time (VRT); and (iv) use of Shigemi tubes.…”